SPAK kinase inhibitors as neuroprotective agents

ABSTRACT

The present disclosure is concerned with N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)benzamide compounds that are capable of inhibiting SPAK kinase function, methods of treating hypoxic brain injuries due to, for example, ischemic stroke. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/588,751, filed on Sep. 30, 2019, which claims the benefit of U.S.Application No. 62/740,336, filed on Oct. 2, 2018, the contents of whichare incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant number VAI01BX002891 awarded by the U.S. Department of Veterans Affairs and grantnumber R01 NS38118 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted May 17, 2021 as a text file named “377590067U3 ST25.txt,” created on May 12, 2021, and having a size of 1,461bytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).

BACKGROUND

Regulation of cellular ion transport is critical for brain waterhomeostasis. Vectorial ion transport across the apical and basolateralmembranes of choroid plexus epithelium (CPe), accompanied by osmotictransport of water, results in daily cerebrospinal fluid (CSF) secretionof >500 cc/day into brain ventricular spaces (Steffensen et al. (2018)Nat Commun 9, 2167). Impaired ionic homeostasis in the CPe can result inhydrocephalus (accumulation of excess CSF in the brain ventricles), asseen in the context of intraventricular hemorrhage (IVH) (Cherian et al.(2004) Brain Pathol 14, 305-311; Strahle et al. (2012) Transl Stroke Res3, 25-38). Coordinated transmembrane influx and efflux of ions and wateris also necessary for cell volume maintenance in neurons and glia.Impaired cell volume homeostasis can result in cytotoxic cell swellingand cerebral edema, as occurs after ischemic stroke (Kahle et al.(2009b) Physiology 24, 257-265; Simard et al. (2007) Lancet Neurol 6,258-268). Ischemic cerebral edema and hydrocephalus are neurosurgicaldiseases, which can treated by decompressive hemicraniectomy orpermanent, catheter-based CSF shunting, respectively (Stochetti and Maas(2014) The New England journal of medicine 371, 972). However, thesemorbid operative procedures have been used for decades with minimalfurther innovation or reduction in failure rates (Wang et al. (1990)Chemotherapy 36, 177-184; Warf et al. (2011) J Neurosurg Pediatr 8,502-508; Wu et al. (2007) Neurosurgery 61, 557-562; discussion 562-553).Thus, novel pharmacological modulators of brain salt and waterhomeostasis are urgently needed to provide a non-surgical alternative tocurrent treatments of these neurological disorders.

The electroneutral cation-Cl⁻ cotransporters (CCCs) are secondary-activeplasmalemmal ion transporters that utilize electrochemically favorabletrans-membrane gradients of Na⁺ and/or K⁺, established by primary activetransport via the ouabain-sensitive Na⁺,K⁺-ATPase, to drive thetransport of Cl⁻ (and K⁺) into or out of cells. The two CCC subtypesinclude the Cl⁻-importing, Na⁺-driven CCCs (NCC, NKCC1, and NKCC2; to bereferred to as “N[K]CCs”), and the Cl⁻-exporting, K⁺-driven CCCs(KCC1-4; the “KCCs”) (Arroyo et al. (2013) Molecular aspects of medicine34, 288-298). These evolutionarily conserved transporters are among themost important mediators of ion transport in multicellular organisms(Gama (2005) Physiological reviews 85, 423-493), with particularimportance in mammalian CNS regulation of ionic and water homeostasis(Gagnon and Delpire (2013) Nature medicine 19, 1524-1528; Kahle et al.(2008) Nat Clin Pract Neurol 4, 490-503). The coordinated regulation ofCCC function is important for cell volume regulation in most braincells, preventing excessive cell swelling or shrinkage in response toosmotic or ischemic stress (Kahle et al. (2015) Trends Mol Med 21,513-523; Zhang et al. (2016) Scientific reports 6, 35986). The centralimportance of CCCs to CSF homeostasis by choroid plexus has beenrecently recognized (Karimy et al. (2017) Nature medicine 23, 997-1003;Steffensen et al. (2018) Nat Commun 9, 2167).

SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidativestress-responsive kinase 1) are closely related Ste20-typeserine-threonine protein kinases considered master regulators of theCCCs (Zhang et al. (2017) Expert Opin Ther Targets 21, 795-804). SPAKand OSR1 are activated by phosphorylation of the regulatory “T-loop”residue (SPAK Thr233 and OSR1 Thr185) by one of the four WNK [“with nolysine” (K)] protein kinases (Moriguchi et al. (2005) The Journal ofbiological chemistry 280, 42685-42693; Vitari et al. (2005) TheBiochemical journal 391, 17-24). WNKs-SPAK/OSR1 protein kinases drivechloride influx by phosphorylation and activation of the Na⁺-driven CCCmembers (NCC, NKCC1, and NKCC2) (Piechotta et al. (2002) The Journal ofbiological chemistry 277, 50812-50819; Richardson et al. (2008) Journalof cell science 121, 675-684; Richardson et al. (2011) Journal of cellscience 124, 789-800) while inhibiting chloride efflux viaphosphorylation and inactivation of KCC1-4 (de Los Heros et al. (2014)The Biochemical journal 458, 559-573; Zhang et al. (2016) Scientificreports 6, 35986). This reciprocal regulation of the Na⁺- and K⁺-drivenCCCs by SPAK/OSR1 ensures that cellular Cl influx and efflux is tightlycoordinated (Arroyo et al. (2013) Molecular aspects of medicine 34,288-298; Kahle et al. (2010) Biochimica et biophysica acta 1802,1150-1158), and is essential for regulation of normal cell volume andepithelial transport in multiple tissues (Damkier et al. (2013)Physiological reviews 93, 1847-1892).

Recent work has highlighted the importance of SPAK-regulatedCCC-mediated ion transport in brain pathologies associated with derangedionic and brain water homeostasis. Experimental ischemic cerebral edemais associated with up-regulated phosphorylation of the SPAK/OSR1 T-loopand NKCC1 (Thr²⁰³/Thr²⁰⁷/Thr²¹²) in both neurons and oligodendrocytes(Begum et al. (2015) Stroke 46, 1956-1965). Mouse germline SPAK knockoutsignificantly reduces ischemia-induced NKCC1 phosphorylation, infarctvolume, axonal demyelination, and cerebral edema following ischemicstroke (Begum et al. (2015) Stroke 46, 1956-1965; Zhao et al. (2017)Journal of cerebral blood flow and metabolism: official journal of theInternational Society of Cerebral Blood Flow and Metabolism 37,550-563). Choroid plexus NKCC1 is an essential mediator of ion transportin the CSF hypersecretory response that drives development ofpost-hemorrhagic hydrocephalus (Karimy et al. (2017) Nature medicine 23,997-1003). The 3.5-fold increase in CSF secretion accompanying thehydrocephalus caused by experimental IVH is associated with similarlyup-regulated phosphorylation of SPAK/OSR1-NKCC1 at the choroid plexusapical membrane (Karimy et al. (2017) Nature medicine 23, 997-1003), thesite of highest SPAK abundance among all epithelial tissues (Piechottaet al. (2003) The Journal of biological chemistry 278, 52848-52856).Genetic inhibition of choroid plexus SPAK by intracerebroventricularsiRNAs normalized CSF secretion rates and reversed post-IVHventriculomegaly (Karimy et al. (2017) Nature medicine 23, 997-1003).Despite the importance of CCCs to CNS physiology, successful developmentof drugs directly targeting CNS CCCs either by inhibiting NKCC1 (Jantzieet al. (2015) Pediatric research 77, 554-562; Kahle and Staley (2008)Neurosurgical focus 25, E22) or activating the KCCs (Gagnon et al.(2013) Cell physiology 304, C693-714) has proven elusive (Cardarelli etal. (2017) Nature medicine 23, 1394-1396). Thus, there remains a needfor compounds that are capable of modulating CCCs via inhibition ofNKCC1 and/or activation of KCCs for reducing ischemic cerebral edemaand/or stimulated CSF recresion.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates toN-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)benzamidecompounds useful in the treatment of conditions or disorders associatedwith a dysregulation of SPAK kinase function including, but not limitedto, hypoxic brain injuries due to, for example, traumatic brain injury,ischemic stroke, carbon monoxide poisoning, drowning, choking,suffocating, or cardiac arrest.

Disclosed are compounds having a structure represented by a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH then R^(2c) is hydrogen, and provided that when R²⁰ is C1-C4 alkylthen at least two of R^(2a), R^(2b), R^(2c), and R^(2d) are nothydrogen, or a pharmaceutically acceptable salt thereof.

Also disclosed are compounds selected from:

or a pharmaceutically acceptable salt thereof.

Also disclosed are pharmaceutical compositions comprising atherapeutically effective amount of at least one disclosed compound anda pharmaceutically acceptable carrier.

Also disclosed are methods for treating a hypoxic brain injury in asubject, the method comprising the step of administering to the subjectan effective amount of at least one compound having a structurerepresented by a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof.

Also disclosed are methods for modifying SPAK kinase function in asubject, the method comprising the step of administering to the subjectan effective amount of a compound having a structure represented by aformula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof.

Also disclosed are methods for modifying SPAK kinase function in atleast one cell, the method comprising the step of administering to thesubject an effective amount of a compound having a structure representedby a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof.

Also disclosed are kits comprising a compound having a structurerepresented by a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof, andone or more of: (a) at least one agent associated with the treatment ofa hypoxic brain injury; (b) instructions for administering the compoundin connection with treating a hypoxic brain injury; and (c) instructionsfor treating a hypoxic brain injury.

Still other objects and advantages of the present disclosure will becomereadily apparent by those skilled in the art from the following detaileddescription, wherein it is shown and described only the preferredembodiments, simply by way of illustration of the best mode. As will berealized, the disclosure is capable of other and different embodiments,and its several details are capable of modifications in various obviousrespects, without departing from the disclosure. Accordingly, thedescription is to be regarded as illustrative in nature and not asrestrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1A shows a representative hybrid design strategy for WNK pathwayinhibitors.

FIG. 1B shows representative data illustrating that ZT-1adoes-dependently inhibits KCC2 Thr906/Thr1007 phosphorylation in cells.

FIG. 2 shows representative concentration-response experiments testingthe effects of Closantel analogs on phosphorylation of KCC2Thr906/Thr1007.

FIG. 3 shows representative data illustrating that ZT-1adose-dependently inhibited KCC2 Thr906/Thr1007 phosphorylation in cells.

FIG. 4A and FIG. 4B show representative data demonstrating the effect ofincreasing concentrations of ZT-1a and staurosporine on SPAK activity atdifferent ATP concentrations.

FIG. 5A and FIG. 5B show representative evidence that SPAK associateswith WNK1 in a manner disrupted by SPAK CCT mutation.

FIG. 6 shows representative evidence that SPAK associates with WNK1 andinteraction is disrupted by ZT-1a and its analogs.

FIG. 7A and FIG. 7B show representative data illustrating the effect ofincreasing concentrations of ZT-1a on SPAK activity in the absence orpresence of wild-type MO25 alpha.

FIG. 8 shows representative data illustrating ⁸⁶Rb⁺ uptake assays in thepresence of ZT-1a.

FIG. 9A and FIG. 9B show a representative in vivo pharmacodynamicanalysis of ZT-1a and Closantel from brain (FIG. 9A) and kidney (FIG.9B), which were administered via subcutaneous injection at the indicateddoses.

FIG. 10A and FIG. 10B show representative data demonstrating thatintracerebroventricular (ICV) delivery of ZT-1a normalizes pathologicalCSF hypersecretion by decreasing choroid plexus CCC phosphorylation.

FIG. 11A shows a representative experimental design of an ischemicstroke study.

FIG. 11B shows representative images and quantification of infarctvolume and hemispheric swelling in TTC-stained coronal sections of mousebrains 24 hrs post-MCAO.

FIG. 12A-C show representative data illustrating that post-strokeadministration of ZT-1a or Closantel does not affect regional cerebralblood flow (rCBF) in ischemic mice.

FIG. 13 shows representative data demonstrating neurological deficitscores, corner tests, and adhesive tape contact and adhesive taperemoval tests of mice treated with vehicle or ZT-1a 1 day before tMCAO(−1) and at days 0, 1, 3, 5, and 7 post-tMCAO.

FIG. 14A and FIG. 14B show representative data demonstrating that ZT-1adecreases ischemia-induced SPAK, NKCC1, and KCC3 phosphorylation in thecerebral cortex post-stroke.

FIG. 15A and FIG. 15B show representative data demonstrating that ZT-1areduces subacute brain gray and white matter injuries after ischemicstroke.

FIG. 16A-C show representative data illustrating that post-strokeadministration of SPAK inhibitor Closantel exhibits dose-dependentneuroprotective effects.

FIG. 17A-C show representative data illustrating that post-strokeadministration of ZT-1a or Closantel does not affect regional cerebralblood flow (rCBF) in ischemic mice.

FIG. 18A-E show representative data illustrating that both male andfemale Angotension II-induced hypertensive mice are responsive to SPAKinhibitor ZT-1a after permanent middle cerebral artery occlusion model(pdMCAO).

FIG. 19A-E show representative data illustrating changes in bloodpressure (BP) and neurological deficit after pdMCAO.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such publication by virtue of prior invention. Further, thedates of publication provided herein may be different from the actualpublication dates, which can require independent confirmation.

A. Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a hypoxic brain injury. The term “patient”includes human and veterinary subjects. In some aspects of the disclosedmethods, the subject has been diagnosed with a need for treatment of ahypoxic brain injury prior to the administering step. In variousaspects, the hypoxic brain injury is due to traumatic brain injury,ischemic stroke, carbon monoxide poisoning, drowning, choking,suffocating, or cardiac arrest.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In various aspects, the term covers anytreatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the condition from occurring in a subject thatcan be predisposed to the condition but has not yet been diagnosed ashaving it; (ii) inhibiting the condition, i.e., arresting itsdevelopment; or (iii) relieving the condition, i.e., causing regressionof the condition. In one aspect, the subject is a mammal such as aprimate, and, in a further aspect, the subject is a human. The term“subject” also includes domesticated animals (e.g., cats, dogs, etc.),livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), andlaboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly,etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit, or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. In some aspects ofthe disclosed methods, the subject has been diagnosed with a need fortreatment of a hypoxic brain injury prior to the administering step. Asused herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder or condition. It is contemplated that theidentification can, in one aspect, be performed by a person differentfrom the person making the diagnosis. It is also contemplated, in afurther aspect, that the administration can be performed by one whosubsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. In various aspects, apreparation can be administered therapeutically; that is, administeredto treat an existing disease or condition. In further various aspects, apreparation can be administered prophylactically; that is, administeredfor prevention of a disease or condition.

The term “treating” refers to relieving the disease, disorder, orcondition, i.e., causing regression of the disease, disorder, and/orcondition. The term “preventing” refers to preventing a disease,disorder, or condition from occurring in a human or an animal that maybe predisposed to the disease, disorder and/or condition, but has notyet been diagnosed as having it; and/or inhibiting the disease,disorder, or condition, i.e., arresting its development.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target (e.g., receptor, cell, etc.), either directly; i.e., byinteracting with the target itself, or indirectly; i.e., by interactingwith another molecule, co-factor, factor, or protein on which theactivity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side effects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In further various aspects, a preparation canbe administered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, “IC₅₀,” is intended to refer to the concentration of asubstance (e.g., a compound or a drug) that is required for 50%inhibition of a biological process, or component of a process, includinga protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, anIC₅₀ can refer to the concentration of a substance that is required for50% inhibition in vivo, as further defined elsewhere herein.

The term “comprising” (and its grammatical variations) as used herein isused in the inclusive sense of “having” or “including” and not in theexclusive sense of “consisting of.”

The compounds according to this disclosure may form prodrugs at hydroxylor amino functionalities using alkoxy, amino acids, etc., groups as theprodrug forming moieties. For instance, the hydroxymethyl position mayform mono-, di- or triphosphates and again these phosphates can formprodrugs. Preparations of such prodrug derivatives are discussed invarious literature sources (examples are: Alexander et al., J. Med.Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO 2000/041531, p. 30).The nitrogen function converted in preparing these derivatives is one(or more) of the nitrogen atoms of a compound of the disclosure.

“Derivatives” of the compounds disclosed herein are pharmaceuticallyacceptable salts, prodrugs, deuterated forms, radio-actively labeledforms, isomers, solvates and combinations thereof. The “combinations”mentioned in this context are refer to derivatives falling within atleast two of the groups: pharmaceutically acceptable salts, prodrugs,deuterated forms, radio-actively labeled forms, isomers, and solvates.Examples of radio-actively labeled forms include compounds labeled withtritium, phosphorous-32, iodine-129, carbon-11, fluorine-18, and thelike.

“Pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The compounds of this disclosure form acidaddition salts with a wide variety of organic and inorganic acids andinclude the physiologically acceptable salts, which are often used inpharmaceutical chemistry. Such salts are also part of this disclosure.Typical inorganic acids used to form such salts include hydrochloric,hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoricacid, and the like. Salts derived from organic acids, such as aliphaticmono- and dicarboxylic acids, phenyl substituted alkanoic acids,hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphaticand aromatic sulfonic acids may also be used. Such pharmaceuticallyacceptable salts thus include acetate, phenylacetate, trifluoroacetate,acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzene-sulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toleunesulfonate,xylenesulfonate, tartarate, and the like.

It is understood that the compounds of the present disclosure relate toall optical isomers and stereo-isomers at the various possible atoms ofthe molecule, unless specified otherwise. Compounds may be separated orprepared as their pure enantiomers or diasteriomers by crystallization,chromatography or synthesis.

The term “leaving group” refers to an atom (or a group of atoms) withelectron withdrawing ability that can be displaced as a stable species,taking with it the bonding electrons. Examples of suitable leavinggroups include sulfonate esters, including triflate, mesylate, tosylate,brosylate, and halides.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. It is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can also be substituted or unsubstituted. The alkyl groupcan be substituted with one or more groups including, but not limitedto, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether,halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.A “lower alkyl” group is an alkyl group containing from one to six(e.g., from one to four) carbon atoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl,sulfo-oxo, or thiol as described herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula (CH₂)_(a)—, where “a” is an integer of from 2to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹-OA² or—OA¹-(OA²)_(a)-OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro,silyl, sulfo-oxo, or thiol, as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onecarbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,norbornenyl, and the like. The term “heterocycloalkenyl” is a type ofcycloalkenyl group as defined above, and is included within the meaningof the term “cycloalkenyl,” where at least one of the carbon atoms ofthe ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group andheterocycloalkenyl group can be substituted or unsubstituted. Thecycloalkenyl group and heterocycloalkenyl group can be substituted withone or more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol asdescribed herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylicacid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl,sulfo-oxo, or thiol, as described herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-basedring composed of at least seven carbon atoms and containing at least onecarbon-carbon triple bound. Examples of cycloalkynyl groups include, butare not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and thelike. The term “heterocycloalkynyl” is a type of cycloalkenyl group asdefined above, and is included within the meaning of the term“cycloalkynyl,” where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkynyl group andheterocycloalkynyl group can be substituted or unsubstituted. Thecycloalkynyl group and heterocycloalkynyl group can be substituted withone or more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol asdescribed herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro,silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is aspecific type of aryl group and is included in the definition of “aryl.”Biaryl refers to two aryl groups that are bound together via a fusedring structure, as in naphthalene, or are attached via one or morecarbon-carbon bonds, as in biphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by theformula NA¹A², where A¹ and A² can be, independently, hydrogen or alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “alkylamino” as used herein is represented by the formula—NH(-alkyl) where alkyl is a described herein. Representative examplesinclude, but are not limited to, methylamino group, ethylamino group,propylamino group, isopropylamino group, butylamino group, isobutylaminogroup, (sec-butyl)amino group, (tert-butyl)amino group, pentylaminogroup, isopentylamino group, (tert-pentyl)amino group, hexylamino group,and the like.

The term “dialkylamino” as used herein is represented by the formula—N(-alkyl)₂ where alkyl is a described herein. Representative examplesinclude, but are not limited to, dimethylamino group, diethylaminogroup, dipropylamino group, diisopropylamino group, dibutylamino group,diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)aminogroup, dipentylamino group, diisopentylamino group, di(tert-pentyl)aminogroup, dihexylamino group, N-ethyl-N-methylamino group,N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein. The term “polyester” as usedherein is represented by the formula —(A¹O(O)C-A²-C(O)O)_(a)— or—(A¹O(O)C-A²-OC(O))_(a)—, where A¹ and A² can be, independently, anoptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and“a” is an integer from 1 to 500. “Polyester” is as the term used todescribe a group that is produced by the reaction between a compoundhaving at least two carboxylic acid groups with a compound having atleast two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group described herein. The term “polyether” as used hereinis represented by the formula —(A¹O-A²O)_(a)—, where A¹ and A² can be,independently, an optionally substituted alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group describedherein and “a” is an integer of from 1 to 500. Examples of polyethergroups include polyethylene oxide, polypropylene oxide, and polybutyleneoxide.

The term “halide” as used herein refers to the halogens fluorine,chlorine, bromine, and iodine.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes pyridinde,pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole,oxazole, thiazole, imidazole, oxazole, including, 1,2,3-oxadiazole,1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole,including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridine, pyridazine,pyrimidine, pyrazine, triazine, including 1,2,4-triazine and1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine,piperidine, piperazine, morpholine, azetidine, tetrahydropyran,tetrahydrofuran, dioxane, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or an optionallysubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or —OS(O)₂OA¹, where A¹ can be hydrogen oran optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.Throughout this specification “S(O)” is a short hand notation for S═O.The term “sulfonyl” is used herein to refer to the sulfo-oxo grouprepresented by the formula S(O)₂A¹, where A¹ can be hydrogen or anoptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “sulfone” as used herein is represented by the formulaA¹S(O)₂A², where A¹ and A² can be, independently, an optionallysubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein. The term“sulfoxide” as used herein is represented by the formula A¹S(O)A², whereA¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. In is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in certain aspects, their recovery,purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(◯); —(CH₂)₀₋₄OR^(◯); —O(CH₂)₀₋₄R^(◯), —O—(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄CH(OR^(◯))₂; —(CH₂)₀₋₄SR^(◯); —(CH₂)₀₋₄Ph, which may besubstituted with R^(◯); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(◯); —CH═CHPh, which may be substituted with R^(◯);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(◯); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(◯))₂; —(CH₂)₀₋₄N(R^(◯))C(O)R^(◯);—N(R^(◯))C(S)R^(◯); —(CH₂)₀₋₄N(R^(◯))C(O)NR^(◯) ₂; —N(R^(◯))C(S)NR^(◯)₂; —(CH₂)₀₋₄N(R^(◯))C(O)OR^(◯); —N(R^(◯))N(R^(◯))C(O)R^(◯);—N(R^(◯))N(R^(◯))C(O)NR^(◯) ₂; —N(R^(◯))N(R^(◯))C(O)OR^(◯);—(CH₂)₀₋₄C(O)R^(◯); —C(S)R^(◯); —(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄C(O)SR^(◯); —(CH₂)₀₋₄C(O)OSiR^(◯) ₃; —(CH₂)₀₋₄OC(O)R^(◯);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(◯); —(CH₂)₀₋₄SC(O)R^(◯); —(CH₂)₀₋₄C(O)NR^(◯)₂; —C(S)NR^(◯) ₂; —C(S)SR^(◯); —SC(S)SR^(◯), —(CH₂)₀₋₄OC(O)NR^(◯) ₂;—C(O)N(OR^(◯))R^(◯); —C(O)C(O)R^(◯); —C(O)CH₂C(O)R^(◯);—C(NOR^(◯))R^(◯); —(CH₂)₀₋₄SSR^(◯); —(CH₂)₀₋₄S(O)₂R^(◯);—(CH₂)₀₋₄S(O)₂OR^(◯); —(CH₂)₀₋₄OS(O)₂R^(◯); —S(O)₂NR^(◯) ₂;—(CH₂)₀₋₄S(O)R^(◯); —N(R^(◯))S(O)₂NR^(◯) ₂; —N(R^(◯))S(O)₂R^(◯);—N(OR^(◯))R^(◯); —C(NH)NR^(◯) ₂; —P(O)₂R^(◯); —P(O)R^(◯) ₂; —OP(O)R^(◯)₂; —OP(O)(OR^(◯))₂; SiR^(◯) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(◯))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(◯))₂, wherein each R^(◯) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(◯), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(◯) (or the ring formed by takingtwo independent occurrences of R^(◯) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●)—(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(◯) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†)2,—C(S)NR^(†)2, —C(NH)NR^(†)2, or —N(R^(†))S(O)₂R^(†); wherein each R^(†)is independently hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

The term “organic residue” defines a carbon-containing residue, i.e., aresidue comprising at least one carbon atom, and includes but is notlimited to the carbon-containing groups, residues, or radicals definedhereinabove. Organic residues can contain various heteroatoms, or bebonded to another molecule through a heteroatom, including oxygen,nitrogen, sulfur, phosphorus, or the like. Examples of organic residuesinclude but are not limited alkyl or substituted alkyls, alkoxy orsubstituted alkoxy, mono or di-substituted amino, amide groups, etc.Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbonatoms, or 1 to 4 carbon atoms. In a further aspect, an organic residuecan comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbonatoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

A very close synonym of the term “residue” is the term “radical,” whichas used in the specification and concluding claims, refers to afragment, group, or substructure of a molecule described herein,regardless of how the molecule is prepared. For example, a2,4-thiazolidinedione radical in a particular compound has thestructure:

regardless of whether thiazolidinedione is used to prepare the compound.In some embodiments the radical (for example an alkyl) can be furthermodified (i.e., substituted alkyl) by having bonded thereto one or more“substituent radicals.” The number of atoms in a given radical is notcritical to the present invention unless it is indicated to the contraryelsewhere herein.

“Organic radicals,” as the term is defined and used herein, contain oneor more carbon atoms. An organic radical can have, for example, 1-26carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms,1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organicradical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbonatoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organicradicals often have hydrogen bound to at least some of the carbon atomsof the organic radical. One example, of an organic radical thatcomprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthylradical. In some embodiments, an organic radical can contain 1-10inorganic heteroatoms bound thereto or therein, including halogens,oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organicradicals include but are not limited to an alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, mono-substituted amino,di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,substituted dialkylcarboxamide, alkyl sulfonyl, alkylsulfinyl,thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl,haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, orsubstituted heterocyclic radicals, wherein the terms are definedelsewhere herein. A few non-limiting examples of organic radicals thatinclude heteroatoms include alkoxy radicals, trifluoromethoxy radicals,acetoxy radicals, dimethylamino radicals and the like.

“Inorganic radicals,” as the term is defined and used herein, contain nocarbon atoms and therefore comprise only atoms other than carbon.Inorganic radicals comprise bonded combinations of atoms selected fromhydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, andhalogens such as fluorine, chlorine, bromine, and iodine, which can bepresent individually or bonded together in their chemically stablecombinations. Inorganic radicals have 10 or fewer, or preferably one tosix or one to four inorganic atoms as listed above bonded together.Examples of inorganic radicals include, but not limited to, amino,hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonlyknown inorganic radicals. The inorganic radicals do not have bondedtherein the metallic elements of the periodic table (such as the alkalimetals, alkaline earth metals, transition metals, lanthanide metals, oractinide metals), although such metal ions can sometimes serve as apharmaceutically acceptable cation for anionic inorganic radicals suchas a sulfate, phosphate, or like anionic inorganic radical. Inorganicradicals do not comprise metalloids elements such as boron, aluminum,gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gaselements, unless otherwise specifically indicated elsewhere herein.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included. During the course of thesynthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the disclosed formulas, it is understoodthat both the (R) and (S) configurations of the chiral carbon, and henceboth enantiomers and mixtures thereof, are embraced within the formula.As is used in the art, when it is desired to specify the absoluteconfiguration about a chiral carbon, one of the bonds to the chiralcarbon can be depicted as a wedge (bonds to atoms above the plane) andthe other can be depicted as a series or wedge of short parallel linesis (bonds to atoms below the plane). The Cahn-Inglod-Prelog system canbe used to assign the (R) or (S) configuration to a chiral carbon.

When the disclosed compounds contain one chiral center, the compoundsexist in two enantiomeric forms. Unless specifically stated to thecontrary, a disclosed compound includes both enantiomers and mixtures ofenantiomers, such as the specific 50:50 mixture referred to as a racemicmixture. The enantiomers can be resolved by methods known to thoseskilled in the art, such as formation of diastereoisomeric salts whichmay be separated, for example, by crystallization (see, CRC Handbook ofOptical Resolutions via Diastereomeric Salt Formation by David Kozma(CRC Press, 2001)); formation of diastereoisomeric derivatives orcomplexes which may be separated, for example, by crystallization,gas-liquid or liquid chromatography; selective reaction of oneenantiomer with an enantiomer-specific reagent, for example enzymaticesterification; or gas-liquid or liquid chromatography in a chiralenvironment, for example on a chiral support for example silica with abound chiral ligand or in the presence of a chiral solvent. It will beappreciated that where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step can liberate the desired enantiomeric form. Alternatively,specific enantiomers can be synthesized by asymmetric synthesis usingoptically active reagents, substrates, catalysts or solvents, or byconverting one enantiomer into the other by asymmetric transformation.

Designation of a specific absolute configuration at a chiral carbon in adisclosed compound is understood to mean that the designatedenantiomeric form of the compounds can be provided in enantiomericexcess (e.e.). Enantiomeric excess, as used herein, is the presence of aparticular enantiomer at greater than 50%, for example, greater than60%, greater than 70%, greater than 75%, greater than 80%, greater than85%, greater than 90%, greater than 95%, greater than 98%, or greaterthan 99%. In one aspect, the designated enantiomer is substantially freefrom the other enantiomer. For example, the “R” forms of the compoundscan be substantially free from the “S” forms of the compounds and are,thus, in enantiomeric excess of the “S” forms. Conversely, “S” forms ofthe compounds can be substantially free of “R” forms of the compoundsand are, thus, in enantiomeric excess of the “R” forms.

When a disclosed compound has two or more chiral carbons, it can havemore than two optical isomers and can exist in diastereoisomeric forms.For example, when there are two chiral carbons, the compound can have upto four optical isomers and two pairs of enantiomers ((S,S)/(R,R) and(R,S)/(S,R)). The pairs of enantiomers (e.g., (S,S)/(R,R)) are mirrorimage stereoisomers of one another. The stereoisomers that are notmirror-images (e.g., (S,S) and (R,S)) are diastereomers. Thediastereoisomeric pairs can be separated by methods known to thoseskilled in the art, for example chromatography or crystallization andthe individual enantiomers within each pair may be separated asdescribed above. Unless otherwise specifically excluded, a disclosedcompound includes each diastereoisomer of such compounds and mixturesthereof.

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labeled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl,respectively. Compounds further comprise prodrugs thereof, andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of this invention. Certainisotopically-labeled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds of the present invention and prodrugsthereof can generally be prepared by carrying out the procedures below,by substituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent.

The compounds described in the invention can be present as a solvate.“Solvates” refers to the compound formed by the interaction of a solventand a solute and includes hydrates. Solvates are usually crystallinesolid adducts containing solvent molecules within the crystal structure,in either stoichiometric or nonstoichiometric proportions. In somecases, the solvent used to prepare the solvate is an aqueous solution,and the solvate is then often referred to as a hydrate. The compoundscan be present as a hydrate, which can be obtained, for example, bycrystallization from a solvent or from aqueous solution. In thisconnection, one, two, three or any arbitrary number of solvate or watermolecules can combine with the compounds according to the invention toform solvates and hydrates. Unless stated to the contrary, the inventionincludes all such possible solvates.

The term “co-crystal” means a physical association of two or moremolecules, which owe their stability through non-covalent interaction.One or more components of this molecular complex provide a stableframework in the crystalline lattice. In certain instances, the guestmolecules are incorporated in the crystalline lattice as anhydrates orsolvates, see e.g. “Crystal Engineering of the Composition ofPharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a NewPath to Improved Medicines?” Almarasson, O., et. al., The Royal Societyof Chemistry, 1889-1896, 2004. Examples of co-crystals includep-toluenesulfonic acid and benzenesulfonic acid.

It is known that chemical substances form solids, which are present indifferent states of order, which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds accordingto the invention can be present in different polymorphic forms, with itbeing possible for particular modifications to be metastable. Unlessstated to the contrary, the invention includes all such possiblepolymorphic forms.

In some aspects, a structure of a compound can be represented by aformula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, R^(n) is understood torepresent five independent substituents, R^(n(a)), R^(n(b)), R^(n(c)),R^(n(d)), R^(n(e)). In each such case, each of the five R^(n) can behydrogen or a recited substituent. By “independent substituents,” it ismeant that each R substituent can be independently defined. For example,if in one instance R^(n(a)) is halogen, then R^(n(b)) is not necessarilyhalogen in that instance.

In some yet further aspects, a structure of a compound can berepresented by a formula:

wherein Ry represents, for example, 0-2 independent substituentsselected from A¹, A², and A³, which is understood to be equivalent tothe groups of formulae:

-   -   wherein Ry represents 0 independent substituents

-   -   wherein Ry represents 1 independent substituent

-   -   wherein Ry represents 2 independent substituents

Again, by “independent substituents,” it is meant that each Rsubstituent can be independently defined. For example, if in oneinstance R^(y1) is A¹, then R^(y2) is not necessarily A¹ in thatinstance.

In some further aspects, a structure of a compound can be represented bya formula,

wherein, for example, Q comprises three substituents independentlyselected from hydrogen and A, which is understood to be equivalent to aformula:

Again, by “independent substituents,” it is meant that each Qsubstituent is independently defined as hydrogen or A, which isunderstood to be equivalent to the groups of formulae:

wherein Q comprises three substituents independently selected from H andA

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. Compounds

In one aspect, disclosed areN-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)benzamidecompounds useful in treating conditions or disorders associated with adysregulation of SPAK kinase function including, but not limited to,hypoxic brain injuries due to, for example, traumatic brain injury,ischemic stroke, carbon monoxide poisoning, drowning, choking,suffocating, or cardiac arrest.

In one aspect, the disclosed compounds exhibit modification of SPAKkinase function. In a further aspect, the disclosed compounds exhibitinhibition of SPAK kinase function.

In one aspect, the disclosed compounds are useful in inhibiting SPAKkinase function in a mammal. In a further aspect, the disclosedcompounds are useful in inhibiting SPAK kinase function in at least onecell.

In one aspect, the disclosed compounds are useful in the treatment ofhypoxic brain injuries, as further described herein.

It is contemplated that each disclosed derivative can be optionallyfurther substituted. It is also contemplated that any one or morederivative can be optionally omitted from the invention. It isunderstood that a disclosed compound can be provided by the disclosedmethods. It is also understood that the disclosed compounds can beemployed in the disclosed methods of using.

1. Structure

In one aspect, disclosed are compounds having a structure represented bya formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH then R^(2c) is hydrogen, and provided that when R²⁰ is C1-C4 alkylthen at least two of R^(2a), R^(2b), R^(2c), and R^(2d) are nothydrogen, or a pharmaceutically acceptable salt thereof.

In one aspect, disclosed are compounds selected from:

or a pharmaceutically acceptable salt thereof.

In a further aspect, the compound has a structure represented by aformula:

In a further aspect, the compound has a structure represented by aformula:

In a further aspect, the compound has a structure represented by aformula:

In a further aspect, the compound is selected from:

-   -   a. R¹Groups

In one aspect, R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b),provided that when R¹ is —OH then R^(2c) is hydrogen. In one aspect, R¹is selected from —OH, —SR²⁰, and —NR^(21a)R^(2b), provided that when R¹is —OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a)and R^(2c) is halogen.

In a further aspect, R¹ is selected from —OH and —SR²⁰. In a stillfurther aspect, R¹ is selected from —OH and —NR^(21a)R^(21b). In yet afurther aspect, R¹ is selected from —SR²⁰ and —NR^(21a)R^(21b).

In a further aspect, R¹ is —OH.

In a further aspect, R¹ is —SR²⁰. In a still further aspect, R¹ is —SH.

In a further aspect, R¹ is NR^(21a)R^(21b).

b. R^(2a), R^(2b), R^(2c), and R^(2d) Groups

In one aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, —NO₂, —CN, —OH, —SH,—NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a further aspect, eachof R^(2a), R^(2b), R^(2c), and R^(2d) is hydrogen.

In a further aspect, each of R^(2b) and R^(2d) is hydrogen. In a stillfurther aspect, each of R^(2a) and R^(2c) is not hydrogen. In yet afurther aspect, at least one of R^(2a), R^(2b), R^(2c), and R^(2d) isnot hydrogen.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, —NO₂, —CN, —OH, —SH,—NH₂, C1-C4 alkyl, and C1-C4 haloalkyl. In a still further aspect, eachof R^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, —F, —Cl, —NO₂, —CN, —OH, —SH, —NH₂, methyl, ethyl, n-propyl,i-propyl, —CH₂Cl, —CH₂CH₂Cl, —CH₂CH₂CH₂Cl, —CH(CH₂Cl)(CH₃), —CH₂F,—CH₂CH₂F, —CH₂CH₂CH₂F, and —CH(CH₂F)(CH₃).

In yet a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, —F, —C1, —NO₂, —CN, —OH, —SH,—NH₂, methyl, ethyl, —CH₂C1, —CH₂CH₂C1, —CH₂F, and —CH₂CH₂F. In an evenfurther aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, —F, —C1, —NO₂, —CN, —OH, —SH,—NH₂, —CH₂C1, and —CH₂F.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, —NO₂, —CN, —OH, —SH, and—NH₂. In a still further aspect, each of R^(2a), R^(2b), R^(2c), andR^(2d) is independently selected from hydrogen, —F, —C1, —NO₂, —CN, —OH,—SH, and —NH₂. In yet a further aspect, each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, —F, —C1, —OH, —SH,and —NH₂. In an even further aspect, each of R^(2a), R^(2b), R^(2c), andR^(2d) is independently selected from hydrogen, —F, —C1, and —OH.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, C1-C4 alkyl, and C1-C4 haloalkyl.In a still further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, —F, —Cl, and —OH.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, C1-C4 alkyl, and C1-C4 haloalkyl.In a still further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, methyl, ethyl, n-propyl, i-propyl,—CH₂Cl, —CH₂CH₂Cl, —CH₂CH₂CH₂Cl, —CH(CH₂Cl)(CH₃), —CH₂F, —CH₂CH₂F,—CH₂CH₂CH₂F, and —CH(CH₂F)(CH₃). In yet a further aspect, each ofR^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, methyl, ethyl, —CH₂Cl, —CH₂CH₂Cl, —CH₂F, and —CH₂CH₂F. In aneven further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, —CH₂Cl, and —CH₂F.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, and C1-C4 alkyl. In astill further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, methyl, ethyl, n-propyl, andi-propyl. In yet a further aspect, each of R^(2a), R^(2b), R^(2c), andR^(2d) is independently selected from hydrogen, halogen, methyl, andethyl. In an even further aspect, each of R^(2a), R^(2b), R^(2c), andR^(2d) is independently selected from hydrogen, halogen, and ethyl. In astill further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, and methyl.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, and C1-C4 haloalkyl. In astill further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, —F, —Cl, —CH₂Cl, —CH₂CH₂Cl,—CH₂CH₂CH₂Cl, —CH(CH₂Cl)(CH₃), —CH₂F, —CH₂CH₂F, —CH₂CH₂CH₂F, and—CH(CH₂F)(CH₃). In yet a further aspect, each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, —F, —Cl, —CH₂Cl,—CH₂CH₂Cl, —CH₂F, and —CH₂CH₂F. In an even further aspect, each ofR^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, —F, —Cl, —CH₂Cl, and —CH₂F.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen and halogen. In a still furtheraspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, —F, —Cl, and —Br. In yet a further aspect, eachof R^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, —F, and —Cl. In an even further aspect, each of R^(2a),R^(2b), R^(2c), and R^(2d) is independently selected from hydrogen and—F. In a still further aspect, each of R^(2a), R^(2b), R^(2c), andR^(2d) is independently selected from hydrogen and —Cl.

c. R²⁰Groups

In one aspect, R²⁰, when present, is selected from hydrogen and C1-C4alkyl, provided that when R²⁰ is C1-C4 alkyl then at least two ofR^(2a), R^(2b), R^(2c), and R^(2d) are not hydrogen. In a furtheraspect, R²⁰, when present, is hydrogen.

In one aspect, R²⁰, when present, is selected from hydrogen and C1-C4alkyl.

In a further aspect, R²⁰, when present, is selected from hydrogen,methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, andt-butyl. In a still further aspect, R²⁰, when present, is selected fromhydrogen, methyl, ethyl, n-propyl, and i-propyl. In yet a furtheraspect, R²⁰, when present, is selected from hydrogen, methyl, and ethyl.In an even further aspect, R²⁰, when present, is selected from hydrogenand ethyl. In a still further aspect, R²⁰, when present, is selectedfrom hydrogen and methyl.

In a further aspect, R²⁰, when present, is selected from methyl, ethyl,n-propyl, propyl, n-butyl, i-butyl, sec-butyl, and t-butyl. In a stillfurther aspect, R²⁰, when present, is selected from methyl, ethyl,n-propyl, and i-propyl. In yet a further aspect, R²⁰, when present, isselected from methyl and ethyl. In an even further aspect, R²⁰, whenpresent, is ethyl. In a still further aspect, R²⁰, when present, ismethyl.

d. R^(21a) and R^(21b) Groups

In one aspect, each of R^(21a) and R^(21b), when present, isindependently selected from hydrogen and C1-C4 alkyl. In a furtheraspect, each of R^(21a) and R^(21b), when present, is hydrogen.

In a further aspect, each of R^(21a) and R^(21b), when present, isindependently selected from hydrogen, methyl, ethyl, n-propyl, i-propyl,n-butyl, i-butyl, sec-butyl, and t-butyl. In a still further aspect,each of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen, methyl, ethyl, n-propyl, and i-propyl. In yet a furtheraspect, each of R^(21a) and R^(21b), when present, is independentlyselected from hydrogen, methyl, and ethyl. In an even further aspect,each of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and ethyl. In a still further aspect, each of R^(21a) andR^(21b), when present, is independently selected from hydrogen andmethyl.

In a further aspect, each of R^(21a) and R^(21b), when present, isindependently selected from methyl, ethyl, n-propyl, i-propyl, n-butyl,i-butyl, sec-butyl, and t-butyl. In a still further aspect, each ofR^(21a) and R^(21b), when present, is independently selected frommethyl, ethyl, n-propyl, and i-propyl. In yet a further aspect, each ofR^(21a) and R^(21b), when present, is independently selected from methyland ethyl. In an even further aspect, each of R^(21a) and R^(21b), whenpresent, is ethyl. In a still further aspect, each of R^(21a) andR^(21b), when present, is methyl.

In a further aspect, R^(21a), when present, is hydrogen, and R^(21b),when present, is C1-C4 alkyl. In a still further aspect, R^(21a), whenpresent, is hydrogen, and R^(21b), when present, is selected frommethyl, ethyl, n-propyl, and i-propyl. In yet a further aspect, R^(21a),when present, is hydrogen, and R^(21b), when present, is selected frommethyl and ethyl. In an even further aspect, R^(21a), when present, ishydrogen, and R^(21b), when present, is ethyl. In a still furtheraspect, R^(21a), when present, is hydrogen, and R^(21b), when present,is methyl.

2. Example Compounds

In one aspect, a compound can be present as one or more of the followingstructures:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be present as one or more of the followingstructures:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be present as one or more of the followingstructures:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be present as one or more of the followingstructures:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be present as one or more of the followingstructures:

or a pharmaceutically acceptable salt thereof

3. Prophetic Compound Examples

The following compound examples are prophetic, and can be prepared usingthe synthesis methods described herein above and other general methodsas needed as would be known to one skilled in the art. It is anticipatedthat the prophetic compounds would be active as inhibitors of SPAKkinase function, and such activity can be determined using the assaymethods described herein.

In one aspect, a compound can be selected from:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be selected from:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be selected from:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be selected from:

or a pharmaceutically acceptable salt thereof.

In one aspect, a compound can be selected from:

or a pharmaceutically acceptable salt thereof.

C. Pharmaceutical Compositions

In one aspect, disclosed are pharmaceutical compositions comprising atleast one disclosed compound and a pharmaceutically acceptable carrier.In a further aspect, a pharmaceutical composition can be providedcomprising a therapeutically effective amount of at least one disclosedcompound. In a still further aspect, a pharmaceutical composition can beprovided comprising a prophylactically effective amount of at least onedisclosed compound. In yet a further aspect, the invention relates topharmaceutical compositions comprising a pharmaceutically acceptablecarrier and a compound, wherein the compound is present in an effectiveamount.

Thus, in one aspect, disclosed are pharmaceutical compositionscomprising a therapeutically effective amount of at least one compoundhaving a structure represented by a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH then R^(2c) is hydrogen, and provided that when R²⁰ is C1-C4 alkylthen at least two of R^(2a), R^(2b), R^(2c), and R^(2d) are nothydrogen, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

Also disclosed are pharmaceutical compositions comprising atherapeutically effective amount of at least one compound selected from:

or a pharmaceutically acceptable salt thereof.

Pharmaceutically acceptable salts of the compounds are conventionalacid-addition salts or base-addition salts that retain the biologicaleffectiveness and properties of the compounds and are formed fromsuitable non-toxic organic or inorganic acids or organic or inorganicbases. Exemplary acid-addition salts include those derived frominorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, andthose derived from organic acids such as p-toluenesulfonic acid,salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citricacid, malic acid, lactic acid, fumaric acid, and the like. Examplebase-addition salts include those derived from ammonium, potassium,sodium and, quaternary ammonium hydroxides, such as for example,tetramethylammonium hydroxide. Chemical modification of a pharmaceuticalcompound into a salt is a known technique to obtain improved physicaland chemical stability, hygroscopicity, flowability and solubility ofcompounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms andDrug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.

The pharmaceutical compositions comprise the compounds in apharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier refers to sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, as well as sterile powders for reconstitutioninto sterile injectable solutions or dispersions just prior to use.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol and the like), carboxymethylcellulose andsuitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. The compounds can beformulated with pharmaceutically acceptable carriers or diluents as wellas any other known adjuvants and excipients in accordance withconventional techniques such as those disclosed in Remington: TheScience and Practice of Pharmacy, 19th Edition, Gennaro, Ed., MackPublishing Co., Easton, Pa., 1995.

In a further aspect, the pharmaceutical composition is administered to amammal. In a still further aspect, the mammal is a human. In an evenfurther aspect, the human is a patient.

In a further aspect, the pharmaceutical composition is administeredfollowing identification of the mammal in need of treatment of cancer.In a still further aspect, the mammal has been diagnosed with a need fortreatment of cancer prior to the administering step.

In a further aspect, the pharmaceutical composition is administeredfollowing identification of the mammal in need of treatment of afibrotic disorder. In a still further aspect, the mammal has beendiagnosed with a need for treatment of a fibrotic disorder prior to theadministering step.

In a further aspect, the pharmaceutical composition is administeredfollowing identification of the mammal in need of immunotherapy. In astill further aspect, the mammal has been diagnosed with a need forimmunotherapy prior to the administering step.

In various aspects, the disclosed pharmaceutical compositions comprisethe disclosed compounds (including pharmaceutically acceptable salt(s)thereof) as an active ingredient, a pharmaceutically acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, topical, andparenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

The choice of carrier will be determined in part by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of the pharmaceutical composition ofthe present invention. The following formulations for oral, aerosol,parenteral, subcutaneous, intravenous, intraarterial, intramuscular,intraperitoneal, intrathecal, rectal, and vaginal administration aremerely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granule; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water, cyclodextrin, dimethylsulfoxide and alcohols, for example, ethanol, benzyl alcohol, propyleneglycol, glycerin, and the polyethylene alcohols including polyethyleneglycol, either with or without the addition of a pharmaceuticallyacceptable surfactant, suspending agent, or emulsifying agent. Capsuleforms can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricants, and inert fillers,such as lactose, sucrose, calcium phosphate, and corn starch. Tabletforms can include one or more of the following: lactose, sucrose,mannitol, corn starch, potato starch, alginic acid, microcrystallinecellulose, acacia, gelatin, guar gum, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, calcium stearate, zincstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acadia, emulsions, and gels containing, theaddition to the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acadia, emulsions, and gels containing, inaddition to the active ingredient, such carriers as are known in theart.

The compounds of the present disclosure alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, and nitrogen. They also may beformulated as pharmaceuticals for non-pressured preparations, such as ina nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol suchas poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid esteror glyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcelluslose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example. dimethyldialkylammoniumhalides, and alkylpyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl β-aminopropionates, and 2-alkylimidazolinequaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically contain from about 0.5% to about25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5% toabout 15% by weight. Suitable surfactants include polyethylene sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol.

Pharmaceutically acceptable excipients are also well-known to those whoare skilled in the art. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical composition of thepresent disclosure. The following methods and excipients are merelyexemplary and are in no way limiting. The pharmaceutically acceptableexcipients preferably do not interfere with the action of the activeingredients and do not cause adverse side-effects. Suitable carriers andexcipients include solvents such as water, alcohol, and propyleneglycol, solid absorbants and diluents, surface active agents, suspendingagent, tableting binders, lubricants, flavors, and coloring agents.

The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.The requirements for effective pharmaceutical carriers for injectablecompositions are well known to those of ordinary skill in the art. SeePharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia,Pa., Banker and Chalmers, Eds., 238-250 (1982) and ASHP Handbook onInjectable Drugs, Toissel, 4^(th) ed., 622-630 (1986).

Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier; as well as creams, emulsions, and gels containing, in additionto the active ingredient, such carriers as are known in the art.

Additionally, formulations suitable for rectal administration may bepresented as suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases. Formulations suitable forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams, or spray formulas containing, in addition to theactive ingredient, such carriers as are known in the art to beappropriate.

One skilled in the art will appreciate that suitable methods ofexogenously administering a compound of the present disclosure to ananimal are available, and, although more than one route can be used toadminister a particular compound, a particular route can provide a moreimmediate and more effective reaction than another route.

As regards these applications, the present method includes theadministration to an animal, particularly a mammal, and moreparticularly a human, of a therapeutically effective amount of thecompound effective in the inhibition of SPAK kinase function. The methodalso includes the administration of a therapeutically effect amount ofthe compound for the treatment of patient having a predisposition forbeing afflicted with a disorder or condition associated with SPAK kinasefunction. The dose administered to an animal, particularly a human, inthe context of the present invention should be sufficient to affect atherapeutic response in the animal over a reasonable timeframe. Oneskilled in the art will recognize that dosage will depend upon a varietyof factors including the condition of the animal, the body weight of theanimal, as well as the severity and stage of the condition.

The total amount of the compound of the present disclosure administeredin a typical treatment is preferably between about 10 mg/kg and about1000 mg/kg of body weight for mice, and between about 100 mg/kg andabout 500 mg/kg of body weight, and more preferably between 200 mg/kgand about 400 mg/kg of body weight for humans per daily dose. This totalamount is typically, but not necessarily, administered as a series ofsmaller doses over a period of about one time per day to about threetimes per day for about 24 months, and preferably over a period of twiceper day for about 12 months.

The size of the dose also will be determined by the route, timing andfrequency of administration as well as the existence, nature and extentof any adverse side effects that might accompany the administration ofthe compound and the desired physiological effect. It will beappreciated by one of skill in the art that various conditions ordisease states, in particular chronic conditions or disease states, mayrequire prolonged treatment involving multiple administrations.

In a further aspect, the composition further comprises at least oneagent associated with the treatment of a hypoxic brain injury. Examplesof an agent associated with the treatment of a hypoxic brain injuryinclude, but are not limited to, thrombolytics, oxygen,antihypertensives, insulin, antipyretics, anticoagulants, andantiplatelet agents. In a still further aspect, the hypoxic brain injuryis due to ischemic stroke.

In a further aspect, the composition further comprises at least oneagent known to have a side effect of increasing the risk of hypoxicbrain injury.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

D. Methods of Making the Compounds

In various aspects, the inventions relates to methods of makingN-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)benzamidecompounds useful in the treatment of conditions or disorders associatedwith a dysregulation of SPAK kinase function including, but not limitedto, hypoxic brain injuries due to, for example, traumatic brain injury,ischemic stroke, carbon monoxide poisoning, drowning, choking,suffocating, or cardiac arrest. Thus, in one aspect, disclosed aremethods of making a disclosed compound.

Compounds according to the present disclosure can, for example, beprepared by the several methods outlined below. A practitioner skilledin the art will understand the appropriate use of protecting groups[see: Greene and Wuts, Protective Groups in Organic Synthesis] and thepreparation of known compounds found in the literature using thestandard methods of organic synthesis. There may come from time to timethe need to rearrange the order of the recommended synthetic steps;however, this will be apparent to the judgment of a chemist skilled inthe art of organic synthesis. The following examples are provided sothat the invention might be more fully understood, are illustrativeonly, and should not be construed as limiting.

In one aspect, the disclosed compounds comprise the products of thesynthetic methods described herein. In a further aspect, the disclosedcompounds comprise a compound produced by a synthetic method describedherein. In a still further aspect, the invention comprises apharmaceutical composition comprising a therapeutically effective amountof the product of the disclosed methods and a pharmaceuticallyacceptable carrier. In a still further aspect, the invention comprises amethod for manufacturing a medicament comprising combining at least onecompound of any of disclosed compounds or at least one product of thedisclosed methods with a pharmaceutically acceptable carrier or diluent.

1. Route I

In one aspect,N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)benzamidecompounds can be prepared as shown below.

Compounds are represented in generic form, with substituents as noted incompound descriptions elsewhere herein and wherein X is a halogen. Amore specific example is set forth below.

In one aspect, compounds of type 1.7, and similar compounds, can beprepared according to reaction Scheme 1B above. Thus, compounds of type1.6 can be prepared by a nucleophilic substitution reaction of anappropriate carboxylic acid, e.g., 1.5 as shown above, and anappropriate activating agent, e.g., thionyl chloride as shown above.Appropriate carboxylic acids and appropriate activating agents arecommercially available or prepared by methods known to one skilled inthe art. Compounds of type 1.7 can be prepared by a coupling reactionbetween an appropriate activated carbonyl compound, e.g., 1.6 as shownabove, and an appropriate amine, e.g., 1.3 as shown above. Appropriateamines are commercially available or prepared by methods known to oneskilled in the art. The coupling reaction is carried out in the presenceof an appropriate solvent, e.g., dioxane, for an appropriate period oftime, e.g., 1 hour, at an appropriate temperature, e.g., 50° C. As canbe appreciated by one skilled in the art, the above reaction provides anexample of a generalized approach wherein compounds similar in structureto the specific reactants above (compounds similar to compounds of type1.1, 1.2, and 1.3), can be substituted in the reaction to providesubstitutedN-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)benzamidecompounds similar to Formula 1.4.

E. Methods of Using the Compounds

The compounds and pharmaceutical compositions of the invention areuseful in treating or controlling conditions or disorders associatedwith a dysregulation of SPAK kinase function including, but not limitedto, hypoxic brain injuries due to, for example, traumatic brain injury,ischemic stroke, carbon monoxide poisoning, drowning, choking,suffocating, or cardiac arrest. In a further aspect, the hypoxic braininjury is due to ischemic stroke or traumatic brain injury. In a stillfurther aspect, the hypoxic brain injury is due to ischemic stroke.

To treat or control the disorder, the compounds and pharmaceuticalcompositions comprising the compounds are administered to a subject inneed thereof, such as a vertebrate, e.g., a mammal, a fish, a bird, areptile, or an amphibian. The subject can be a human, non-human primate,horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.The term does not denote a particular age or sex. Thus, adult andnewborn subjects, as well as fetuses, whether male or female, areintended to be covered. The subject is preferably a mammal, such as ahuman. Prior to administering the compounds or compositions, the subjectcan be diagnosed with a need for treatment of a cancer, immunedysfunction, or of a fibrotic disorder.

The compounds or compositions can be administered to the subjectaccording to any method. Such methods are well known to those skilled inthe art and include, but are not limited to, oral administration,transdermal administration, administration by inhalation, nasaladministration, topical administration, intravaginal administration,ophthalmic administration, intraaural administration, intracerebraladministration, rectal administration, sublingual administration, buccaladministration and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. A preparation can beadministered therapeutically; that is, administered to treat an existingdisease or condition. A preparation can also be administeredprophylactically; that is, administered for prevention of a hypoxicbrain injury.

The therapeutically effective amount or dosage of the compound can varywithin wide limits. Such a dosage is adjusted to the individualrequirements in each particular case including the specific compound(s)being administered, the route of administration, the condition beingtreated, as well as the patient being treated. In general, in the caseof oral or parenteral administration to adult humans weighingapproximately 70 Kg or more, a daily dosage of about 10 mg to about10,000 mg, preferably from about 200 mg to about 1,000 mg, should beappropriate, although the upper limit may be exceeded. The daily dosagecan be administered as a single dose or in divided doses, or forparenteral administration, as a continuous infusion. Single dosecompositions can contain such amounts or submultiples thereof of thecompound or composition to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days.

1. Treatment Methods

The compounds disclosed herein are useful for treating or controllingconditions or disorders associated with a dysregulation of SPAK kinasefunction including, but not limited to, hypoxic brain injuries due to,for example, traumatic brain injury, ischemic stroke, carbon monoxidepoisoning, drowning, choking, suffocating, or cardiac arrest. Thus,provided is a method comprising administering a therapeuticallyeffective amount of a composition comprising a disclosed compound to asubject. In a further aspect, the method can be a method for treating ahypoxic brain injury.

a. Treating a Hypoxic Brain Injury

In one aspect, disclosed are methods of treating a hypoxic brain injuryassociated with SPAK kinase function in a mammal, the method comprisingthe step of administering to the mammal an effective amount of at leastone disclosed compound, or a pharmaceutically acceptable salt thereof.

Thus, in one aspect, disclosed are methods for treating a hypoxic braininjury in a subject, the method comprising the step of administering tothe subject an effective amount of at least one compound having astructure represented by a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof.

A hypoxic brain injury can be caused by or due to, for example,traumatic brain injury, ischemic stroke, carbon monoxide poisoning,drowning, choking, suffocating, or cardiac arrest.

In a further aspect, the hypoxic brain injury is caused by or due to atraumatic brain injury or ischemic stroke. In a still further aspect,the hypoxic brain injury is caused by or due to ischemic stroke.

In a further aspect, the subject has been diagnosed with a need fortreatment of a hypoxic brain injury prior to the administering step.

In a further aspect, the subject is a mammal. In a still further aspect,the mammal is a human.

In a further aspect, the effective amount is a therapeutically effectiveamount. In a still further aspect, the effective amount is aprophylactically effective amount.

In a further aspect, the hypoxic brain injury is associated withdysregulation of SPAK kinase.

In a further aspect, the method further comprises the step ofidentifying a subject in need of treatment of a hypoxic brain injury.

In a further aspect, the method further comprises the step ofadministering a therapeutically effective amount of at least one agentassociated with the treatment of a hypoxic brain injury. Examples ofagents associated with the treatment of hypoxic brain injuries include,but are not limited to, thrombolytics (e.g., anistrplase, reteplase,streptokinase, kabikinase, tenecteplase, rokinase), oxygen,antihypertensives (e.g., ACE inhibitors such as benazepril, captopril,enalapril, fosinopril, Lisinopril, moexipril, perindopril, andquinapril; angiotensin II receptor blockers such as azilsartan,candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan,and valsartan; beta blockers such as acebutolol, atenolol, bisoprolol,metoprolol, nadolol, nebivolol, and propranolol; calcium channelblockers such as amlodipine, diltiazem, felodipine, isradipine,nicardipine, nifedipine, nisoldipine, and verapamil; direct renininhibitors such as aliskiren; and diuretics), insulin, antipyretics(e.g., salicylates such as acetylsalicylic acid, choline salicylate,magnesium salicylate, and sodium salicylate; acetaminophen; andnonsteroidal anti-inflammatory drugs such as ibuprofen, naproxen, andketoprofen), anticoagulants (e.g., heparin, warfarin, rivaroxaban,dabigatran, apixaban, edoxaban, enoxaparin, fondaparinux), andantiplatelet agents (e.g., clopidogrel, ticagrelor, prasugrel,dipyridamole, dipyridamole/aspirin, ticlodipine, and eptfibatide).

In a further aspect, the at least one compound and the at least oneagent are administered sequentially. In a still further aspect, the atleast one compound and the at least one agent are administeredsimultaneously.

In a further aspect, the at least one compound and the at least oneagent are co-formulated. In a still further aspect, the at least onecompound and the at least one agent are co-packaged.

2. Methods for Modifying SPAK Kinase Function in a Subject

In one aspect, disclosed are methods for modifying SPAK kinase functionin a subject, the method comprising the step of administering to thesubject an effective amount of a compound having a structure representedby a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof.

In one aspect, disclosed are methods of modifying SPAK kinase functionin a mammal, the method comprising the step of administering to themammal a therapeutically effective amount of at least one disclosedcompound, or a pharmaceutically acceptable salt thereof.

In a further aspect, the compound exhibits inhibition of SPAK kinasefunction. In a still further aspect, the compound exhibits a decrease inSPAK kinase function.

In a further aspect, the subject is a mammal. In a still further aspect,the subject is a human.

In a further aspect, the subject has been diagnosed with a hypoxic braininjury prior to the administering step. In a still further aspect, themethod further comprises the step of identifying a subject in need oftreatment of a hypoxic brain injury prior to the administering step.

In a further aspect, the subject has been diagnosed with a need formodifying SPAK kinase function prior to the administering step. In afurther aspect, the subject has been diagnosed with a need forinhibiting SPAK kinase function prior to the administering step.

In a further aspect, the subject has been diagnosed with a need fortreatment of the disorder associated with SPAK kinase function prior tothe administering step. In a still further aspect, the subject has beendiagnosed with a need for treatment of the disorder associated with SPAKkinase dysfunction prior to the administering step. In yet a furtheraspect, the method further comprises the step of identifying a subjectin need of treatment of a disorder associated with SPAK kinase function.In an even further aspect, the method further comprises the step ofidentifying a subject in need of treatment of a disorder associated withSPAK kinase dysfunction.

3. Methods for Modifying SPAK Kinase Function in at Least One Cell

In one aspect, disclosed are methods for modifying SPAK kinase functionin at least one cell, the method comprising the step of contacting theat least one cell with an effective amount of at least one disclosedcompound, or a pharmaceutically acceptable salt thereof.

In one aspect, disclosed are methods for modifying SPAK kinase functionin at least one cell, the method comprising the step of administering tothe subject an effective amount of a compound having a structurerepresented by a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof.

In a further aspect, modifying is decreasing. In a still further aspect,modifying is inhibiting.

In a further aspect, the cell is mammalian. In a still further aspect,the cell is human. In yet a further aspect, the cell has been isolatedfrom a mammal prior to the contacting step.

In a further aspect, contacting is via administration to a mammal.

In a further aspect, the subject has been diagnosed with a hypoxic braininjury prior to the administering step. In a still further aspect, themethod further comprises the step of identifying a subject in need oftreatment of a hypoxic brain injury prior to the administering step.

In a further aspect, the subject has been diagnosed with a need formodifying SPAK kinase function prior to the administering step. In afurther aspect, the subject has been diagnosed with a need forinhibiting SPAK kinase function prior to the administering step.

In a further aspect, the subject has been diagnosed with a need fortreatment of the disorder associated with SPAK kinase function prior tothe administering step. In a still further aspect, the subject has beendiagnosed with a need for treatment of the disorder associated with SPAKkinase dysfunction prior to the administering step. In yet a furtheraspect, the method further comprises the step of identifying a subjectin need of treatment of a disorder associated with SPAK kinase function.In an even further aspect, the method further comprises the step ofidentifying a subject in need of treatment of a disorder associated withSPAK kinase dysfunction.

4. Use of Compounds

In one aspect, the invention relates to the use of a disclosed compoundor a product of a disclosed method. In a further aspect, a use relatesto the manufacture of a medicament for the treatment of a hypoxic braininjury in a mammal.

Also provided are the uses of the disclosed compounds and products. Inone aspect, the invention relates to use of at least one disclosedcompound; or a pharmaceutically acceptable salt, hydrate, solvate, orpolymorph thereof. In a further aspect, the compound used is a productof a disclosed method of making.

In a further aspect, the use relates to a process for preparing apharmaceutical composition comprising a therapeutically effective amountof a disclosed compound or a product of a disclosed method of making, ora pharmaceutically acceptable salt, solvate, or polymorph thereof, foruse as a medicament.

In a further aspect, the use relates to a process for preparing apharmaceutical composition comprising a therapeutically effective amountof a disclosed compound or a product of a disclosed method of making, ora pharmaceutically acceptable salt, solvate, or polymorph thereof,wherein a pharmaceutically acceptable carrier is intimately mixed with atherapeutically effective amount of the compound or the product of adisclosed method of making.

In various aspects, the use relates to a treatment of a disorder orcondition in a mammal. Also disclosed is the use of a compound formodification of SPAK kinase function. In one aspect, the use ischaracterized in that the mammal is a human. In one aspect, the use ischaracterized in that the disorder or condition is a hypoxic braininjury. In one aspect, the hypoxic brain injury is due to ischemicstroke.

In a further aspect, the use relates to the manufacture of a medicamentfor the treatment of a hypoxic brain injury in a mammal.

In a further aspect, the use relates to modulation of SPAK kinasefunction in a mammal. In a further aspect, the use relates to inhibitionof SPAK kinase function in a mammal. In a still further aspect, the userelates to modulation of SPAK kinase function in a cell. In yet afurther aspect, the mammal is a human.

It is understood that the disclosed uses can be employed in connectionwith the disclosed compounds, products of disclosed methods of making,methods, compositions, and kits. In a further aspect, the inventionrelates to the use of a disclosed compound or a disclosed product in themanufacture of a medicament for the treatment of a hypoxic brain injuryin a mammal. In a further aspect, the hypoxic brain injury is due toischemic stroke.

5. Manufacture of a Medicament

In one aspect, the invention relates to a method for the manufacture ofa medicament for treating a disorder or condition associated with SPAKkinase function in a mammal, the method comprising combining atherapeutically effective amount of a disclosed compound or product of adisclosed method with a pharmaceutically acceptable carrier or diluent.

As regards these applications, the present method includes theadministration to an animal, particularly a mammal, and moreparticularly a human, of a therapeutically effective amount of thecompound effective in the inhibition of SPAK kinase function. The doseadministered to an animal, particularly a human, in the context of thepresent invention should be sufficient to affect a therapeutic responsein the animal over a reasonable timeframe. One skilled in the art willrecognize that dosage will depend upon a variety of factors including,for example, the condition of the animal and the body weight of theanimal.

The total amount of the compound of the present disclosure administeredin a typical treatment is preferably between about 10 mg/kg and about1000 mg/kg of body weight for mice, and between about 100 mg/kg andabout 500 mg/kg of body weight, and more preferably between 200 mg/kgand about 400 mg/kg of body weight for humans per daily dose. This totalamount is typically, but not necessarily, administered as a series ofsmaller doses over a period of about one time per day to about threetimes per day for about 24 months, and preferably over a period of twiceper day for about 12 months.

The size of the dose also will be determined by the route, timing andfrequency of administration as well as the existence, nature and extentof any adverse side effects that might accompany the administration ofthe compound and the desired physiological effect. It will beappreciated by one of skill in the art that various conditions ordisease states, in particular chronic conditions or disease states, mayrequire prolonged treatment involving multiple administrations.

Thus, in one aspect, the invention relates to the manufacture of amedicament comprising combining a disclosed compound or a product of adisclosed method of making, or a pharmaceutically acceptable salt,solvate, or polymorph thereof, with a pharmaceutically acceptablecarrier or diluent.

6. Kits

In one aspect, disclosed are kits comprising a compound having astructure represented by a formula:

wherein R¹ is selected from —OH, —SR²⁰, and —NR^(21a)R^(21b); whereinR²⁰, when present, is selected from hydrogen and C1-C4 alkyl; whereineach of R^(21a) and R^(21b), when present, is independently selectedfrom hydrogen and C1-C4 alkyl; wherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, halogen, —NO₂, —CN,—OH, —SH, —NH₂, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4hydroxyalkyl, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 aminoalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino, provided that when R¹ is—OH and each of R^(2b) and R^(2d) is hydrogen then neither of R^(2a) andR^(2c) is halogen, or a pharmaceutically acceptable salt thereof, andone or more of: (a) at least one agent associated with the treatment ofa hypoxic brain injury; (b) instructions for administering the compoundin connection with treating a hypoxic brain injury; and (c) instructionsfor treating a hypoxic brain injury.

Hypoxic brain injuries can be caused by or due to, for example,traumatic brain injury, ischemic stroke, carbon monoxide poisoning,drowning, choking, suffocating, or cardiac arrest. In a further aspect,the hypoxic brain injury is due to traumatic brain injury or ischemicstroke. In a still further aspect, the hypoxic brain injury is due toischemic stroke.

Examples of agents associated with the treatment of hypoxic braininjuries include, but are not limited to, thrombolytics (e.g.,anistrplase, reteplase, streptokinase, kabikinase, tenecteplase,rokinase), oxygen, antihypertensives (e.g., ACE inhibitors such asbenazepril, captopril, enalapril, fosinopril, Lisinopril, moexipril,perindopril, and quinapril; angiotensin II receptor blockers such asazilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan,telmisartan, and valsartan; beta blockers such as acebutolol, atenolol,bisoprolol, metoprolol, nadolol, nebivolol, and propranolol; calciumchannel blockers such as amlodipine, diltiazem, felodipine, isradipine,nicardipine, nifedipine, nisoldipine, and verapamil; direct renininhibitors such as aliskiren; and diuretics), insulin, antipyretics(e.g., salicylates such as acetylsalicylic acid, choline salicylate,magnesium salicylate, and sodium salicylate; acetaminophen; andnonsteroidal anti-inflammatory drugs such as ibuprofen, naproxen, andketoprofen), anticoagulants (e.g., heparin, warfarin, rivaroxaban,dabigatran, apixaban, edoxaban, enoxaparin, fondaparinux), andantiplatelet agents (e.g., clopidogrel, ticagrelor, prasugrel,dipyridamole, dipyridamole/aspirin, ticlodipine, and eptfibatide). Thus,in a further aspect, the agent associated with the treatment of ahypoxic brain injury is selected from a thrombolytic, oxygen, anantihypertensive, insulin, an antipyretic, an anticoagulant, and anantiplatelet agent.

In a further aspect, the at least one compound and the at least oneagent are co-formulated. In a further aspect, the at least one compoundand the at least one agent are co-packaged.

The kits can also comprise compounds and/or products co-packaged,co-formulated, and/or co-delivered with other components. For example, adrug manufacturer, a drug reseller, a physician, a compounding shop, ora pharmacist can provide a kit comprising a disclosed compound and/orproduct and another component for delivery to a patient.

It is understood that the disclosed kits can be prepared from thedisclosed compounds, products, and pharmaceutical compositions. It isalso understood that the disclosed kits can be employed in connectionwith the disclosed methods of using.

The foregoing description illustrates and describes the disclosure.Additionally, the disclosure shows and describes only the preferredembodiments but, as mentioned above, it is to be understood that it iscapable to use in various other combinations, modifications, andenvironments and is capable of changes or modifications within the scopeof the invention concepts as expressed herein, commensurate with theabove teachings and/or the skill or knowledge of the relevant art. Theembodiments described herein above are further intended to explain bestmodes known by applicant and to enable others skilled in the art toutilize the disclosure in such, or other, embodiments and with thevarious modifications required by the particular applications or usesthereof. Accordingly, the description is not intended to limit theinvention to the form disclosed herein. Also, it is intended to theappended claims be construed to include alternative embodiments.

All publications and patent applications cited in this specification areherein incorporated by reference, and for any and all purposes, as ifeach individual publication or patent application were specifically andindividually indicated to be incorporated by reference. In the event ofan inconsistency between the present disclosure and any publications orpatent application incorporated herein by reference, the presentdisclosure controls.

F. Examples

Here, the development of a “dual” CCC modulator (NKCC1 inhibitor/KCCactivator),5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide(“ZT-1a”) that potently and selectively inhibits SPAK kinase—the CCCmaster regulator—is disclosed. ZT-1a-mediated SPAK inhibition leads toreduced cellular ion influx and stimulated cellular Cl⁻ extrusion bysimultaneous reduction of the activating phosphorylation of NKCC1 andthe inhibitory phosphorylation of the KCCs. Intracerebroventriculardelivery of ZT-1a normalizes CSF hypersecretion in hemorrhagichydrocephalus by decreasing SPAK-mediated phosphorylation of NKCC1 andKCC4 in choroid plexus. Systemic ZT-1a administration after experimentalischemic stroke attenuates cerebral infarction and edema and improvesneurological outcomes. Without wishing to be bound by theory, theseresults suggest ZT-1a holds promise as an effective kinase-cotransportermodulator capable of restoring brain water homeostasis and improvingneurological function in vivo.

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representative.

1. General Chemistry Experimental Methods

Reagents and solvents were obtained from commercial sources and wereused without further purification. Reactions were monitored by thinlayer chromatography (TLC) on glass plates coated with silica gel withfluorescent indicator. Compounds were purified either by chromatographyon silica gel or by preparative high performance liquid chromatography(HPLC). Silica gel chromatography was performed on the Teledyne ISCOCombiFlash system (RF200) eluting with petroleum ether/ethyl acetate(PE/EA) or dichloromethane/methanol (DCM/MeOH). Preparative HPLC wasconducted on the Waters autopurification system consisting of a 2767sample manager, a 2545 binary gradient module, a 2489 UV detector, and a3100 mass detector. NMR spectra were recorded on a Bruker UltrashieldPlus-600 (600 MHz) spectrometer and chemical shifts are reported in δ(ppm). ¹H chemical shifts are reported as s (singlet), d (doublet), dd(doublet of doublet), t (triplet), q (quartet), m (multiplet), and brs(broad singlet) and are referenced to the residual solvent signal:DMSO-d₆ (2.50). ¹³C spectra are referenced to the residual solventsignal: DMSO-d₆ (39.52). High Resolution Mass Spectra were obtained on aThermo Fisher Scientific LTQ FTICR-MS. The purity of all testedcompounds was >95% by HPLC.

2. General Synthesis of Compounds 1a-1j

Reagents and conditions: a) SOCl₂, reflux; b)2-(4-amino-2-chloro-5-methylphenyl)-2-(4-chlorophenyl) acetonitrile,Dioxane, 50° C., 1 h.

a. Preparation of 5-chloro-2-hydroxybenzoyl Chloride (I-1a)

The mixture of 5-chloro-2-hydroxy-benzoic acid (103.5 mg, 0.6 mmol) in1.5 mL of thionyl chloride was refluxed at 80° C. for 2 h. The resultingsolution was cooled down to room temperature and the excess thionylchloride was removed under vacuum to afford I-1a as a gummy yellowsolid. Then I-1a was directly used in next step.

b.5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxy-benzamide(1a; ZT-1a)

To a stirred solution of2-(4-amino-2-chloro-5-methylphenyl)-2-(4-chlorophenyl)acetonitrile (87.3mg, 0.3 mmol) in 1,4-dioxane (4 mL) was added I-1a (85.9 mg, 0.45 mmol)at room temperature, and the resulting mixture was heated at 50° C. for1 h. Then the reaction was cooled to room temperature and extracted withethyl acetate (20 mL). The organic extract was washed with brine (20mL), dried over anhydrous MgSO₄, and concentrated in vacuum. The crudesolid was purified by column chromatography on silica gel(dichloromethane/petroleum ether=3/1) to afford 1a as white crystals,with a yield of 60.9 mg, 45%. ¹H NMR (600 MHz, DMSO-d₆) δ 12.20 (s, 1H),10.48 (s, 1H), 8.29 (s, 1H), 7.96 (d, J=2.8 Hz, 1H), 7.53-7.48 (m, 4H),7.40 (d, J=8.5 Hz, 2H), 7.07 (d, J=8.8 Hz, 1H), 5.89 (s, 1H), 2.34 (s,3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 163.6, 156.1, 138.4, 134.3, 133.8,133.4, 131.9, 130.1, 130.0, 129.6, 129.4, 128.9, 123.9, 123.4, 120.2,119.5, 38.5, 17.7. MS (ESI) m/z: 445[M+H]⁺. HRMS (ESI) calculated forC₂₂H₁₄Cl₃N₂O₂ [M−H]⁻, 443.0121; found, 443.0115.

c.N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxy-benzamide(1b)

Compound 1b (42.7 mg, 34.6%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 9.90 (s,1H), 9.79 (s, 1H), 7.63 (s, 1H), 7.52-7.51 (m, 2H), 7.51-7.50 (m, 1H),7.43-7.42 (m, 1H), 7.41-7.39 (m, 2H), 7.35 (dd, J=3.7, 1.8 Hz, 1H), 7.33(d, J=7.8 Hz, 1H), 7.02-6.98 (m, 1H), 6.03 (s, 1H), 2.29 (s, 3H). ¹³CNMR (150 MHz, DMSO-d₆) δ 166.1, 157.9, 138.6, 136.0, 134.3, 133.8,133.4, 132.0, 130.3, 130.0, 130.0, 129.6, 129.5, 127.6, 119.5, 119.3,118.7, 115.1, 38.6, 18.0. MS (ESI) m/z: 411[M+H]⁺. HRMS (ESI) calculatedfor C22H₁₇Cl₂N₂O₂ [M+H]⁺, 411.0662; found, 411.0662.

d.N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-mercapto-benzamide(1c)

Compound 1c (20 mg, 31.2%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 8.10-8.01(m, 1H), 7.96 (dt, J=7.8, 1.0 Hz, 1H), 7.77 (ddd, J=8.4, 7.2, 1.3 Hz,1H), 7.69 (s, 2H), 7.54-7.50 (m, 3H), 7.49-7.44 (m, 2H), 6.12 (s, 1H),2.19 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 163.6, 141.4, 137.4, 136.3,133.7, 133.1, 133.0, 132.4, 132.1, 130.7, 129.7, 129.6, 129.1, 126.1,125.8, 122.9, 122.0, 118.7, 38.1, 17.1. MS (ESI) m/z: 427 [M+H]⁺. HRMS(ESI) calculated for C22H₁₅Cl₂N₂OS [M−H]⁻, 425.0282; found, 425.0277.

e.2-amino-n-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-benzamide(1d)

To a stirred solution of R¹ (41.0 mg, 0.1 mmol) in acetic acid (1 mL)was added metallic iron powder (16.8 mg, 0.3 mmol) at room temperature.The reaction mixture was heated to 56° C. for 1.5 hours and then cooledto room temperature. The solvent was removed under reduced pressure andextracted with ethyl acetate. The organic layer was washed with brine,dried over anhydrous MgSO₄, and concentrated under reduced pressure. Thecrude solid was purified by column chromatography on silica gel(dichloromethane/petroleum ether=3/1) to afford 1d as white crystals,with a yield of 20.6 mg, 50.2%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.73 (s,1H), 7.69 (d, J=7.1 Hz, 1H), 7.61 (s, 1H), 7.52 (s, 1H), 7.51 (d, J=2.4Hz, 2H), 7.41 (d, J=8.5 Hz, 2H), 7.22 (dd, J=11.2, 4.1 Hz, 1H), 6.77 (d,J=8.3 Hz, 1H), 6.61 (t, J=7.5 Hz, 1H), 6.03 (s, 1H), 2.28 (s, 3H). ¹³CNMR (150 MHz, DMSO-d₆) δ 168.3, 150.4, 138.8, 134.3, 133.8, 133.4,132.9, 131.9, 130.0, 129.6, 129.5, 129.3, 127.6, 119.6, 117.1, 115.4,114.7, 110.0, 38.5, 18.0. MS (ESI) m/z: 410 [M+H]⁺. HRMS (ESI)calculated for C₂₂H₁₈Cl₂N₃O [M+H]⁺, 410.0821; found, 410.0821.

f.N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-nitrobenzamide(1e)

Compound R¹ (78.5 mg, 59.4%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s,1H), 8.19 (d, J=8.1 Hz, 1H), 7.91 (t, J=7.4 Hz, 1H), 7.84 (d, J=7.3 Hz,1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.52 (d, J=1.2 Hz, 2H), 7.50 (s, 1H),7.41 (d, J=8.4 Hz, 2H), 6.05 (s, 1H), 2.30 (s, 3H). ¹³C NMR (150 MHz,DMSO-d₆) δ 165.3, 146.8, 137.7, 134.7, 134.2, 133.4, 132.8, 132.7,132.2, 131.5, 130.3, 130.0, 129.9, 129.7, 129.6, 126.6, 124.8, 119.5,38.6, 17.8. MS (ESI) m/z: 440[M+H]t HRMS (ESI) calculated forC₂₂H₁₄Cl₂N₃₀₃ [M−H]⁻, 438.0412; found, 438.0407.

g.3,5-dichloro-n-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxy-benzamide(1f)

Compound 1f (78.5 mg, 59.4%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 10.63 (s,1H), 8.05 (d, J=2.5 Hz, 1H), 7.84 (d, J=2.5 Hz, 1H), 7.76 (d, J=2.7 Hz,1H), 7.75-7.73 (m, 1H), 7.55 (s, 1H), 7.52-7.48 (m, 2H), 7.43-7.39 (m,2H), 6.04 (s, 1H), 2.29 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.0,136.7, 133.7, 133.1, 133.0, 133.0, 131.6, 130.5, 129.6, 129.3, 129.2,128.3, 126.9, 126.8, 122.9, 122.5, 119.0, 118.8, 38.1, 17.3. MS (ESI)m/z: 479 [M+H]⁺. HRMS (ESI) calculated for C22H₁₃C14N₂O₂ [M−H]⁻,476.9731; found, 476.9726.

h.N-(5-chloro-4-o-chlorophenyl)(cyano)methyl)-2-methylphenyl)-5-fluoro-2-hydroxy-benzamide(1g)

Compound 1g (78.5 mg, 59.4%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 11.97 (s,1H), 10.55 (s, 1H), 8.33 (s, 1H), 7.73 (dd, J=9.6, 3.3 Hz, 1H),7.53-7.50 (m, 2H), 7.50-7.48 (m, 1H), 7.43-7.38 (m, 2H), 7.33 (ddd,J=8.9, 7.8, 3.3 Hz, 1H), 6.01 (s, 1H), 2.34 (s, 3H). MS (ESI) m/z:429[M+H]⁺. HRMS (ESI) calculated for C₂₂H₁₄Cl₂FN₂O₂ [M−H]⁻, 427.0416;found, 427.0411.

i.N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-3-fluoro-2-hydroxy-benzamide(1h)

Compound 1h (78.5 mg, 59.4%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 12.24 (s,1H), 10.61 (s, 1H), 8.11 (s, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.53 (s, 1H),7.52-7.49 (m, 2H), 7.46 (ddd, J=10.8, 8.1, 1.5 Hz, 1H), 7.42-7.39 (m,2H), 6.99 (td, J=8.1, 4.8 Hz, 1H), 6.03 (s, 1H), 2.33 (s, 3H). MS (ESI)m/z: 429[M+H]⁺. HRMS (ESI) calculated for C₂₂H₁₄Cl₂FN₂O₂ [M−H]⁻,427.0416; found, 427.0411.

j.N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2,3-dihydroxy-benzamide(1I)

Compound 1i (78.5 mg, 59.4%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 11.26 (s,1H), 10.56 (s, 1H), 9.77 (s, 1H), 8.19 (s, 1H), 7.50 (dd, J=6.7, 4.7 Hz,3H), 7.46 (dd, J=8.1, 1.5 Hz, 1H), 7.41-7.37 (m, 2H), 7.01 (dd, J=7.8,1.6 Hz, 1H), 6.86-6.77 (m, 1H), 6.02 (s, 1H), 2.31 (s, 3H). ¹³C NMR (150MHz, DMSO-d₆) δ 166.0, 147.0, 146.6, 138.5, 134.3, 133.4, 131.9, 130.1,130.0, 130.0 129.6, 128.9, 124.1, 120.1, 119.6, 119.6, 119.5, 118.5,38.5, 17.8. MS (ESI) m/z: 427[M+H]⁺. HRMS (ESI) calculated forC₂₂H₁₅Cl₂N₂O₃ [M−H]⁻, 425.0460; found, 425.0454.

k.5-chloro-n-(5-chloro-4-((4-chlorophenyl)(cyanomethyl)-2-methylphenyl)-2-methoxy-benzamide(1j)

Compound 1j (78.5 mg, 59.4%) was prepared according to the methodsdescribed in the synthesis of 1a. ¹H NMR (600 MHz, DMSO-d₆) δ 10.01 (s,1H), 8.16 (s, 1H), 7.85 (d, J=2.8 Hz, 1H), 7.63 (dd, J=8.9, 2.8 Hz, 1H),7.52 (s, 1H), 7.52-7.51 (m, 1H), 7.51-7.49 (m, 1H), 7.40 (d, J=8.5 Hz,1H), 7.31 (d, J=8.9 Hz, 1H), 6.02 (s, 1H), 4.00 (s, 3H), 2.35 (s, 3H).¹³C NMR (150 MHz, DMSO-d₆) δ 162.8, 156.3, 138.3, 134.3, 133.4, 133.1,131.9, 130.4, 130.0, 130.0, 129.9, 129.6, 129.1, 125.3, 124.5, 123.9,119.6, 115.1, 57.3, 38.5, 17.6. MS (ESI) m/z: 459 [M+H]⁺. HRMS (ESI)calculated for C₂₂H₁₅Cl₂N₂O₃ [M−H]⁻, 457.0277; found, 457.0272.

3. General Biology Experimental Methods

Tissue culture reagents were from Life Technologies. P81phosphocellulose paper was from Whatman and [—³²P]-ATP was from PerkinElmer. CATCHtide was synthesised by Pepceuticals. Protein G sepharosewas from Amersham. DNA constructs used for transfection were purifiedfrom Escherichia coli DH5a using Qiagen or Invitrogen plasmid Maxi kitsaccording to the manufacturer's protocol. All DNA constructs wereverified by DNA sequencing.

a. Plasmids, Protein Expression and Purification

DNA clones were from the Division of Signal Transduction Therapy(University of Dundee). The SPAK proteins were expressed in E. Coli andpurified as described previously (Zhang et al. (2015) Human moleculargenetics 24: 4545-4558).

b. SPAK Kinase Assays and IC₅₀ Determination

SPAK kinase assays employing CATCHtide (cation chloride co-transporterpeptide substrate). Peptide kinase assays, performed in triplicate, wereset up in a total volume of 50 μl containing 0.5 μg GST-[T233E]SPAKkinase (˜5 nM at ˜10% purity) in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 10mM MgCl₂, 300 μM CATCHtide (RRHYYYDTHTNTYYLRTFGHNTRR; SEQ ID NO:1), 0.1μM [γ-32P]ATP (˜500 cpm/pmol) and the indicated concentrations ofinhibitor dissolved in DMSO. After incubation for 30 min at 30° C.,reactions were terminated by spotting 40 μl of the reaction mix onto P81phosphocellulose paper and immersion in 50 mM phosphoric acid. Thepapers were washed extensively and the incorporation of [γ-³²P]ATP intoCATCHtide was quantified by Cerenkov counting. IC⁵⁰ values werecalculated with GraphPad Prism using non-linear regression analysis.

c. Fluorescence Polarization

Fluorescence polarization measurements were performed at 25° C. withpurified SPAK proteins in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl and 2 mMDTT. The concentration of SPAK proteins was determined by measuringtheir absorbance at 280 nm and calculated using the molar absorptioncoefficient determined by the ProtParam Online tool (Gasteiger et al.,2001). All peptides (SEEGKPQLVGRFQVTSSK [EP4543] and SEEGKPQLVGAFQVTSSK[EP4544]; SEQ ID NO:2 and SEQ ID NO:3, respectively) contained anN-terminal linker required for conjugating to the Lumio Greenfluorophore (CCPGCCGGGG; SEQ ID NO:4) and were initially re-suspended in50 mM ammonium bicarbonate, pH 8. Peptide labelling was achieved byincubating 10 nM of each peptide in a 0.5 ml reaction mixture of 20 μMLumio Green in 25 mM Tris/HCl, pH 7.5, 200 mM NaCl and 5 mM2-mercaptoethanol. Reactions were left to proceed in the dark for 2 h.The peptides were dialysed for 4 h into 25 mM Tris/HCl, pH 7.5, 200 mMNaCl and 5 mM 2-mercaptoethanol using a Micro DispoDIALYZER with a 100Da molecular-mass cut-off (Harvard Apparatus), and then for another 12 hwith changed buffer. For fluorescence polarization, mixtures were set upcontaining the indicated concentration of protein, 10 nMLumio-Green-labelled peptide in a final volume of 30 μl. All individualbindings were performed in duplicate with at least 12 data points percurve. Fluorescence polarization was measured using a BMG PheraStarplate reader, with an excitation wavelength of 485 nm and an emissionwavelength of 538 nm, and measurements were corrected to the fluorescentprobe alone. Data analysis and graphing were then performed in GraphPadPrism7; a one-site specific binding model was assumed (Y=Bmax*X/[Kd+X])and the fitted dissociation constant computed. All experimental bindingswere repeated at least twice.

d. Pharmacodynamic (PD) Study

Male C57BL/6j wild-type mice (6 weeks old) were purchased from CharlesRiver Laboratories Edinburgh UK. ZT-1a was dissolved in DMSO (Sigma)solution and administered by subcutaneous injection into wild type maleC57BL/6 mice at doses of 0, 3, 10, 30, 50 and 100 mg/kg. 50 mg/kgClosantel (1) was injected as a comparative control. Age-matchedSPAK^(502A/502A) knock-in mice were also used as comparative controls(Zhang et al. (2015) Human molecular genetics 24: 4545-455). Controlmice were treated with an equal volume of DMSO solution. One hour afteradministration, mice were sacrificed by cervical dislocation, and kidneyand brain tissues were rapidly dissected and snap-frozen in liquidnitrogen. The SPAK^(502A/502A) knock-in mouse was established andmaintained under specific pathogen-free conditions at the University ofDundee (UK) as described in our recent study. Animal experiments andbreeding were approved by the University of Dundee ethical committee andperformed under a U.K. Home Office project license, in accordance withthe Animals (Scientific Procedures) Act 1986, the Policy on the Care,Welfare and Treatment of Animals, and regulations set by the Universityof Dundee and the U.K. Home Office.

e. Cell Culture, Transfections and Stimulations

HEK293 (human embryonic kidney 293) cells were cultured on10-cm-diameter dishes in DMEM supplemented with 10% (v/v) foetal bovineserum, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.HEK293 cells were transfected with a mixture of 20 μl of 1 mg/mlpolyethylenimine (Polysciences) and 5-10 μg of plasmid DNA as describedpreviously (Durocher et al., 2002). 36 hours post-transfection cellswere stimulated with either control isotonic or hypotonic medium for aperiod of 30 minutes. Cells were lysed in 0.3 mL of ice-cold lysisbuffer/dish, lysates were clarified by centrifugation at 4° C. for 15minutes at 26,000 g and the supernatant aliquots were frozen in liquidnitrogen and stored at −20° C. Protein concentrations were determinedusing the Bradford method. Cells were treated with the indicatedconcentrations of the SPAK/OSR1 inhibitors. Isotonic buffer was 135 mMNaCl, 5 mM KCl, 0.5 mM CaCl₂), 0.5 mM MgCl₂, 0.5 mM Na₂HPO₄, 0.5 mMNa₂SO₄ and 15 mM HEPES (pH 7.5). Hypotonic low chloride buffer was 67.5mM sodium-gluconate, 2.5 mM potassium-gluconate, 0.25 mM CaCl₂), 0.25 mMMgCl₂, 0.5 mM Na₂HPO₄, 0.5 mM Na₂SO₄ and 7.5 mM HEPES (pH 7.5).

f. Immunoblotting and Phospho-Antibody Immunoprecipitation

Protein samples (40 μg) were boiled in sample buffer for 5 min, resolvedby 7.5% sodium dodecyl sulfate polyacrylamide-gel electrophoresis andelectrotransferred onto a polyvinylidene difluoride membrane aspreviously described (Bhuiyan et al. (2017) Journal of cerebralbloodflow and metabolism: official journal of the International Societyof Cerebral Blood Flow and Metabolism 37: 2780-2794; de Los Heros et al.(2014) The Biochemical journal 458: 559-573; Zhu et al. (2014) Molecularcancer 13: 31). The membranes were incubated for 30 min with TBST(Tris-buffered saline, 0.05% Tween-20) containing 5% (w/v) skim milk.The blots were then washed six times with TBST and incubated for 1 hourat room temperature with secondary HRP-conjugated antibodies diluted5000-fold in 5% (w/v) skim milk in TBST. After repeating the washingsteps, signals were detected with enhanced chemiluminescence reagent.Immunoblots were developed using a film automatic processor (SRX-101;Konica Minolta Medical) and films were scanned at 600 dpi (PowerLook1000; UMAX). Figures were generated using Photoshop/Illustrator (Adobe).The densities of bands were measured with ImageJ. For phospho-antibodyimmunoprecipitation, KCC isoforms were immunoprecipitated from indicatedcell extracts. 2 mg of the indicated clarified cell extract were mixedwith 15 μg of the indicated phospho-specific KCC antibody conjugated to15 μl of protein-G-Sepharose, in the added presence of 20 μs of thedephosphorylated form of the phosphopeptide antigen, and incubated 2hours at 4° C. with gentle shaking. Immunoprecipitates were washed threetimes with 1 mL of lysis buffer containing 0.15 M NaCl and twice with 1ml of buffer A. Bound proteins were eluted with 1×LDS sample buffer.

g. ⁸⁶RB⁺ Uptake Assay in HEK293 Cells

HEK293 cells were plated at 50-60% confluence of in 12-well plates(2.4-cm-diameter per/well) and transfected with wild-type or variousmutant forms of full-length flag-tagged human KCCs. Each well of HEK293cells was transfected with 2.5 μL of 1 mg/mL polyethylenimine and 1 μgof plasmid DNA. The ⁸⁶Rb⁺-uptake assay was performed on cells at 36hours post-transfection. Culture medium was aspirated from the wells andreplaced with either isotonic or hypotonic medium for 15 min at 37° C.,then for a further 15 min with stimulating medium containing additional1 mM ouabain and 0.1 mM bumetanide, to prevent ⁸⁶Rb⁺ uptake via theNKCC1 cotransporter. This stimulating medium was then removed andreplaced with isotonic medium plus inhibitors containing 2 μCi/mL ⁸⁶Rb⁺for 10 min at 37° C., following which cells were rapidly washed threetimes with the respective ice-cold non-radioactive medium. The cellswere lysed in 300 μL of ice-cold lysis buffer and ⁸⁶Rb⁺ uptake wasquantitated by liquid scintillation counting (PerkinElmer).

h. Model of Post-Hemorrhagic Hydrocephalus

All animal experiments were approved by the Institutional Animal Careand Use Committee (IACUC) of the Yale University, and in accordance withthe guidelines and regulations in the NIH Guide for the Care and Use ofLaboratory Animals. Male Wistar rats (Harlan, Indianapolis, Ind., USA),age 8 weeks (220-230 g), were anesthetized (60 mg/kg ketamine plus 7.5mg/kg xylazine, IP) and allowed to breathe room air spontaneously. Bodytemperature was maintained at 37±1° C. (Harvard Apparatus, Holliston,Mass., USA) throughout the course of the experiments. PHH was modeledusing a modified protocol based on previously described methods (Karimyet al. (2015) Nature medicine 23: 997-1003; Simard et al. (2011) TranslStroke Res 2: 227-231). Briefly, in an anesthetized animal, the tailartery was aseptically cannulated using a flexible catheter (PE-20)pre-loaded with heparinized saline. The rat was then mounted in astereotactic apparatus (Stoelting Co., Wood Dale, Ill.), a midline scalpincision was made to expose the skull and a 1 mm burr hole was madeusing a high-speed drill over the right lateral ventricle (coordinates,x=−0.8, y=−1.7 mm relative to bregma). Approximately 200 μL of blood wasdrawn from the tail artery catheter and loaded into a 500 μL syringe(Hamilton, Reno, Nev.), which was mounted to the stereotactic frame.Under stereotactic guidance, 50 μL of freshly collected autologousblood, free from anticoagulants, was infused into the right lateralventricle (coordinates, x=−0.8, y=−1.7, z=−4.5 mm relative to bregma),over the course of 5 minutes, and the 26-gauge needle is held in placefor an additional 20 minutes to prevent backflow of blood upon needleremoval.

i. Quantitation of Rates of CSF Production and IntracerebroventricularDrug Administration

Rates of CSF production were measured using the method we recentlypublished (Karimy et al. (2015) Nature medicine 23: 997-1003). Briefly,anesthetized rats were mounted in a stereotactic apparatus and a 1.3 mmburr hole was made over the left lateral ventricle (coordinates, x=−0.8,y=+1.7 relative to bregma). Over the right lateral ventricle, an Alzetbrain infusion cannula (#1; Durect, Cupertino, Calif.) was mounted withone spacer to adjust to −4.5 mm depth. The cannula was then connected toa 1 mL syringe loaded with either ZT-1a or vehicle solution (see below)via PE-20 catheter tubing to a syringe infusion apparatus (Pump elite11; Harvard Apparatus). Next, the rat's head was rotated on the ear-bars90°, nose-down, and the suboccipital muscles were dissected to thecisterna magna to expose the atlanto-occipital ligament. The ligament ispunctured and a 23-gauge flexible catheter (PE-20) was advanced 5 mmthrough the foramen of Magendie to the 4th ventricle. Sterile, moleculargrade mineral oil (100 μL; Sigma Aldrich, St. Louis, Mo.) was infusedinto the 4th ventricle to occlude the aqueduct of Slyvius, therebycreating a closed system of CSF circulation. With the rat in the sameposition, a glass capillary tube (cat #CV8010-300; borosilicate; OD, 1mm; ID, 0.8 mm; length, 30 cm; VitroCom, Mountain Lakes, N.J.) isadvanced through the burr hole into the left lateral ventricle. Thevolume (V) of CSF that forms at a given timepoint is calculated as: V(mm³)=π·r²·d, where r is the radius of the capillary tube and d is thedistance CSF traveled within the capillary. The rate of CSF formation(μL/min) can be calculated from the slope of the volume-timerelationship. A baseline rate of spontaneous CSF secretion (no druginfusion) was calculated over 30 minutes prior to drug infusion, thencompared to the calculated rate of CSF formation following 30 minutes ofZT-1a infusion.

ZT-1a solution preparation. Intraventricular infusion solutions weremade using sterile artificial cerebrospinal fluid (aCSF), composed asfollows (in mM): sodium, 150; potassium, 3.0; calcium, 1.4; magnesium,0.8; phosphorus, 1.0; chlorine, 155, pH 7.19 (Tocris, Bristol, UK), witha calculated osmolarity of 311.2 mOsmC/1 (Karimy et al., 2015; Karimy etal., 2017). ZT-1a was dissolved in CSF using DMSO as a co-solvent andtitrated to pH 9 (10 mmol, 1% DMSO, pH 9). As a control, a vehiclesolution of aCSF (1% DMSO, pH 9) omitting ZT-1a was prepared and tested.

j. Tissue Harvest and Choroid Plexus Isolation.

Rats were euthanized via overdose of pentobarbital (Euthasol)administered IP and transcardially perfused with ice-cold normal saline.The brain was rapidly isolated and then placed in an ice-cold salinebath, after which the choroid plexus was carefully dissected undermagnification using sharp forceps. Approximately 3 mg of choroid plexustissue was harvested from each brain, which was then collected into a1.5 mL tube and flash frozen in liquid nitrogen for storage.

k. Middle Cerebral Artery Occlusion Model

Focal cerebral ischemia was induced by transient occlusion of the leftmiddle cerebral artery (MCA) for 50 min as described previously (Bhuiyanet al. (2017) Journal of cerebral blood flow and metabolism: officialjournal of the International Society of Cerebral Blood Flow andMetabolism 37: 2780-2794; Zhao et al. (2017) Journal of cerebral bloodflow and metabolism: official journal of the International Society ofCerebral Blood Flow and Metabolism 37: 550-563). Under an operatingmicroscope, the left common carotid artery was exposed through a midlineincision. Two branches of the external carotid artery (ECA), occipitaland superior thyroid arteries, were isolated and coagulated. The ECA wasdissected further distally and permanently ligated. The internal carotidartery (ICA) was isolated and carefully separated from the adjacentvagus nerve. A 12 mm length of silicon-coated nylon filament (size 6-0,native diameter 0.11 mm; diameter with coating 0.21+/−0.02 mm; coatinglength 5-6 mm; Doccol Corporation, Sharon, Mass.) was introduced intothe ECA lumen through a puncture. The silk suture around the ECA stumpwas tightened around the intraluminal nylon suture to prevent bleeding.The nylon suture was then gently advanced from the ECA to the ICA lumenuntil mild resistance was felt (˜9 mm). For reperfusion, the suture waswithdrawn 50 min after MCAO to restore blood flow. Body temperature wasmaintained for the duration of the experiment between 36.5° C.-37° C.with a small animal heating pad (Kent Scientific).

l. Cerebral Blood Flow Measurement

Cerebral blood flow was measured using a two-dimensional laser specklecontrast analysis system (PeriCam PSI High Resolution with PIMSoft,Perimed) (Begum et al. (2017) Glia, DOI: 10.1002/glia.23232; Chung-YangYeh (2017) J Neurosci 37: 5648-5658). Isoflurand-anesthetized mice werehead-fixed using stereotaxic equipment during imaging. The skin wasretracted to expose the intact skull. Images were taken at 19frames/second with averaging. Average signal intensity was taken from afixed size (0.5 mm²) regions of interest drawn over the parietal boneplate on the ipsi- and contra-lateral sides. Percent perfusion valueswere taken in comparison to the mean values of the pre-ischemicipsilateral side. Five consecutive images at each time period per animalwere averaged for analysis.

m. Brain Infarction Volume and Hemispheric Swelling Measurements

At 24 hours reperfusion, mice were anesthetized with 5% isoflurane andthen decapitated. Coronal brain tissue slices (2 mm) were stained for 20minutes at 37° C. with 1% 2,3,5-triphenyltetrazolium chloridemonohydrate (TTC; Sigma, St Louis, Mo.) in PBS solution. Infarctionvolume was calculated with correction for edema using ImageJ software asdescribed (Swanson et al. (1990) Journal of cerebral blood flow andmetabolism: official journal of the International Society of CerebralBlood Flow and Metabolism 10: 290-293). The extent of hemisphericswelling was calculated using the following equation: swelling (%contralateral side)=[(volume of ipsilateral hemisphere−volume ofcontralateral hemisphere)/volume of contralateral hemisphere]×100(Bhuiyan et al. (2017) Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism 37: 2780-2794).

n. Neurological Deficit Score

A neurological deficit grading system (Bhuiyan et al. (2017) Journal ofcerebral blood flow and metabolism: official journal of theInternational Society of Cerebral Blood Flow and Metabolism 37:2780-2794; Zhao et al. (2017) Journal of cerebral blood flow andmetabolism: official journal of the International Society of CerebralBlood Flow and Metabolism 37: 550-563) was used to evaluate neurologicaldeficit at 0, 1, 3, 5, and 7 days after tMCAO. The scores were: 0, noobservable deficit; 1, forelimb flexion; 2, forelimb flexion anddecreased resistance to lateral push; 3, forelimb flexion, decreasedresistance to lateral push, and unilateral circling; 4, forelimb flexionand partial or complete lack of ambulation.

o. Corner Test

Corner test was used to assess MCAO-induced sensorimotor abnormalitiesas described previously (Zhao et al. (2017) Journal of cerebral bloodflow and metabolism: official journal of the International Society ofCerebral Blood Flow and Metabolism 37: 550-563). In brief, the cornertest apparatus consists of two cardboards (30×20×1 cm each) placedtogether at a 30° angle to form a narrow alley. The mouse was placedbetween the two angled boards facing the corner. When exiting thecorner, uninjured mice turned left or right randomly. After tMCAO,animals with unilateral brain damage displayed an asymmetry in cornerturning. The numbers of left and right turns of each mouse during 10trials were recorded, and turning movements that were not part of arearing movement were not scored. Preoperative training was carriedtwice per day for three days prior to operation. Postoperatively,animals were tested on day 1, 3, 5, and 7.

p. Adhesive Removal Test

An adhesive removal test was used to measure somatosensory deficits asdescribed previously (Begum et al. (2017) Glia, DOI: 10.1002/glia.23232;Zhao et al. (2017) Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism 37: 550-563). In brief, two small pieces of adhesive tape (4mm×3 mm) were attached to the forepaws in an alternating sequence andwith equal pressure by the experimenter before each trial. Animals werereleased into a testing cage, and the time of contact and removal of theadhesive patch were recorded. Contact was recorded when either shakingof the paw or mouth contact occurred. The trial ended after the adhesivepatch was removed or after 2 min had elapsed. Preoperative training wascarried twice per day for three days prior to operation.Postoperatively, animals were tested on day 1, 3, 5, and 7.

q. DTI of Ex-Vivo Brains

Seven days post-reperfusion, mice were anesthetized with 5% isoflurane,transcardially perfused with 4% paraformaldehyde (PFA) and decapitated(Zhao et al. (2017) Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism 37: 550-563). For ex-vivo MRI, brains were maintained withinthe skull to avoid anatomical deformation. After overnight post-fixationin 4% PFA, heads were stored in PBS solution at 4° C. MM was performedat 500 MHz using a Bruker AV3HD 11.7 T/89 mm vertical bore small animalMRI scanner, equipped with a 25-mm quadrature RF coil and Paravision 6.0(Bruker Biospin, Billerica, Mass.). Following positioning and pilotscans, a DTI data set covering the entire brain was collected using amultislice spin echo sequence with 3 reference and 30 noncollineardiffusion-weighted images with the following parameters: TE/TR=22/5000ms, 4 averages, matrix size=192×192 reconstructed to 256×256, field ofview=17.3×17.3 mm, 33 axial slices, slice thickness=0.5 mm, b-value=1200s/mm2, and Δ/δ=10/5 ms.

DTI data were analyzed with DSI Studio (Zhao et al. (2017) Journal ofcerebral blood flow and metabolism: official journal of theInternational Society of Cerebral Blood Flow and Metabolism 37:550-563). In a blinded manner, region of interests (ROIs) were manuallydrawn for the ipsilateral (IL) and contralateral (CL) cortex, striatum,corpus callosum (CC), and external capsule (EC). Values for thefractional anisotropy (FA), afferent diffusion coefficient (ADC), anddiffusivity were calculated for the entire volume of each ROI.

r. Preparation of Brain Membrane and Cytosolic Protein Fractions

Brain homogenates were prepared as previously described (Bhuiyan et al.(2017) Journal of cerebral blood flow and metabolism: official journalof the International Society of Cerebral Blood Flow and Metabolism 37:2780-2794). Mice were anesthetized with 5% isoflurane vaporized in N₂₀and 02 (3:2), then decapitated. The contralateral (CL) and ipsilateral(IL) brain tissues were dissected in five volumes of cold homogenizationbuffer (0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4, protease andphosphatase inhibitor cocktail (Pierce). Brain tissues were gentlyhomogenized with a tissue grinder (Kontes, Vineland, N J, USA) for 10strokes in homogenization buffer. The homogenized samples werecentrifuged at 1000×g at 4° C. for 10 min. The supernatant (51) wascollected and centrifuged at 200,000×g for 30 min in a Beckman Optima™XL-80k Ultracentrifuge. The cytosolic fraction [supernatant (S2)] andcrude membrane pellet were collected. The pellet was re-suspended in thehomogenization buffer. Protein content in both membrane and cytosolicfractions was determined by the standard bicinchoninic acid method.

s. Animal Preparation

All animal experiments were approved by the University of PittsburghInstitutional Animal Care and Use Committee and performed in accordancewith the National Institutes of Health Guide for the Care and Use ofLaboratory Animals. The manuscript adheres to the ARRIVE guidelines forreporting animal experiments. Eleven to 16-week old C57BL/6J mice (bothmale and female) were used in the study (Jackson laboratories, BarHarbor, Me.).

t. Focal Ischemic Stroke and Reperfusion with Transient Middle CerebralArtery Occlusion

Focal ischemic stroke was induced by transient occlusion of the leftmiddle cerebral artery (MCA) for 50 min in normotensive mice asdescribed previously (Huang et al. (2019) Stroke. 50:1021-1025). Underan operating microscope, the left common carotid artery was exposedthrough a midline incision. Two branches of the external carotid artery(ECA), occipital and superior thyroid arteries, were isolated andcoagulated. The ECA was dissected further distally and permanentlyligated. The internal carotid artery (ICA) was isolated and carefullyseparated from the adjacent vagus nerve. A 12 mm length ofsilicon-coated nylon filament (size 6-0, native diameter 0.11 mm;diameter with coating 0.21+/−0.02 mm; coating length 5-6 mm; DoccolCorporation, Sharon, Mass.) was introduced into the ECA lumen through apuncture. The silk suture around the ECA stump was tightened around theintraluminal nylon suture to prevent bleeding. The nylon suture was thengently advanced from the ECA to the ICA lumen until mild resistance wasfelt (˜9 mm). For reperfusion, the suture was withdrawn 50 min afterMCAO to restore blood flow. Body temperature was maintained for theduration of the experiment between 36.5° C.-37° C. with a small animalheating pad (Kent Scientific).

u. ZT-1a Pharmacokinetic (PK) Properties in Sham Control and tMCAOIschemic Stroke Normotensive Mice

ZT-1a content in brain homogenates and plasma was assayed at theUniversity of Pittsburgh Small Molecule Biomarker Core, using liquidchromatography-tandem mass spectrometry (LC-MS/MS). C57BL/6j mice(normotensive male, 11 weeks old) at 3 hr post Sham surgery or tMCAOwere intraperitoneally injected with 5 mg/kg ZT-1a. At 2 hr postinjection, blood was collected into heparin-treated EP tubes by acardiac puncture. After a brief cardiac perfusion with ice-cold normalsaline, brain tissues (contralateral and ipsilateral hemispheres) werecollected. Plasma samples were prepared by centrifugation of whole bloodfor 10 min at 1,500 g using a refrigerated centrifuge. Brain tissues andplasma samples were diluted, and protein was precipitated withacetonitrile prior to detection of ZT-1a with a triple quad massspectrometer. Glyburide was used as the internal standard. Samples werethen injected by autosampler and ZT-1a was eluted from a Waters AcquityUPLC BEH C18, 1.7 um, 2.1×100 mm reversed-phase column with a water(with 0.1% formic acid) and acetonitrile (with 0.1% formic acid)gradient. Detection and quantitation of ZT-1a were achieved in thepositive mode with a Thermo Fisher TSQ Quantum Ultra mass spectrometerinterfaced via an electrospray ionization (ESI) probe with the WaterUPLC Acquity solvent delivery system.

v. Ang II-Mediated Hypertension (HTN) and Bp Measurement

C57BL/6J mice received subcutaneous (s.c.) infusion of either saline orAngII via osmotic minipumps (model 1004, Alzet) at a rate of 1000ng/kg/min for 14 days. BP was measured in awake mice by a tail-cuffmethod (Kent Scientific) as described previously (Huang et al. (2019)Stroke. 50:1021-1025).

w. Focal Cerebral Ischemia with Permanent Middle Cerebral ArteryOcclusion

Saline-infused control and Ang II-mediated HTN mice were subsequentlyunderwent permanent occlusion of the distal branches of the middlecerebral artery (pdMCAO) as described previously (Huang et al. (2019)Stroke. 50:1021-1025). Under isoflurane anesthesia, a skin incision atthe midline of the neck was made and the left CCA was exposed andoccluded by ligation and the skin was sutured. Another skin incision (1cm) was made between the left eye and the ear using fine operationscissors. The temporal muscle was identified and detached from the skullin its apical and dorsal part without totally removing the muscle by theforceps. The MCA below the transparent skull in the rostral part of thetemporal area, dorsal to the retro-orbital sinus was identified. If theMCA bifurcation was not visible (due to an anatomical normal variation),the most rostral vessel was identified. The skull above the MCA branchwas thinned out with the drill until it had a thin and translucenttexture. The artery, proximal and distal to the MCA bifurcation, wascoagulated with the electrocoagulation forceps (in a bipolar mode at 7W). The temporal muscle was relocated to its position and the burr holewas covered with wax and the skin wound was sutured and infiltrated withanalgesia bupivacaine (100 μl 0.25%) topically. The animal was placed ina cage and monitored for recovery from anesthesia. Body temperature wasmaintained for the duration of the experiment between 36.5-37° C. with asmall animal heating pad (Kent Scientific). Sham-operated AngII-mediatedhypertensive mice underwent identical surgical procedures but were notsubjected to vessel coagulation.

x. Drug Treatment in HTN Mice

Ang II-mediated HTN mice were randomly assigned to receive eithervehicle (Veh, 100% DMSO, 2 ml/kg body weight/day) or ZT-1a (5.0 mg/kgbody weight/day), administered via intraperitoneal injection (i.p.) withan initial half dose at 3-hours and the second half dose at 8-hoursreperfusion.

y. Brain Infarction Volume and Hemispheric Swelling Measurements in HTNMice

Cerebral infarction and hemisphere swelling were assessed at 24 hoursreperfusion as described previously (Swanson etmal. (1990) J Cereb BloodFlow Metab. 10:290-293; Bhuiyan et al. (2017) J Cereb Blood Flow Metab.37:2780-2794).

z. Neurological Function Tests in HTN Mice

Foot fault, cylinder test, and adhesive tape removal tests were used toassess pdMCAO-induced somatosensory and motor deficits in HTN mice in ablinded manner as described previously (Bhuiyan et al. (2017)J CerebBlood Flow Metab. 37:2780-2794; Begum et al. (2018) Glia. 66).

aa. Statistical Analysis

Animal subjects were randomly assigned into different studies andsurgical procedures, and data analyses were performed by investigatorsblinded to experimental conditions. The number of animals studied was80% powered to detect 20% changes with a (2-sided)=0.05. Data wereexpressed as mean±SEM. Statistical significance was determined bystudent's t-test, or one-way ANOVA using the Tukey's post-hoc test incase of multiple comparisons (GraphPad Prism 6.0, San Diego, Calif.,USA). Neurological deficit score was analyzed by the non-parametricMann-Whitney test. A probability value <0.05 was consideredstatistically significant.

4. Restoration of Brain Water Homeostasis and Neurological Function Viaa Novel Kinase-Cotransporter Modulator

a. Identification of ZT-1a: A Novel and Potent Non-ATP-Competitive SPAKInhibitor.

To identify pharmacological modulators of SPAK kinase, a new focusedchemical library derived from the previously identified SPAK inhibitorsClosantel (Kikuchi et al. (2015) Journal of the American Society ofNephrology: JASN 26, 1525-1536), Rafoxanide (Alamri et al. (2017)ChemMedChem 12, 639-645), and STOCK1S-14279 (Kikuchi et al. (2015)Journal of the American Society of Nephrology: JASN 26, 1525-1536) wasdesigned and synthesized (FIG. 1A). This “scaffold-hybrid” strategy hasproven useful for the successful development of kinase inhibitors withhigh selectivity (Deng et al. (2011) Nature chemical biology 7, 203-205;Deng et al. (2013) Eur J Med Chem 70, 758-767). Closantel and Rafoxanidetarget the allosteric site on the C-terminal domains of SPAK and OSR1,rather than the highly-conserved ATP-binding pocket of kinases, therebyinhibiting kinase activity in a non-ATP competitive manner (Alamri etal. (2017) ChemMedChem 12, 639-645)). Iterative rounds of medicinalchemistry optimization led to identification of “ZT-1a”[5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide]as a highly selective SPAK inhibitor (Table 1 and FIG. 1A).

Referring to FIG. 1A, a representative hybrid design strategy for WNKpathway inhibitors is shown.

TABLE 1

SPAK NKCC1 KCC2 KCC2 pSer373 pThr203 pThr906 pThr1007 CompoundSubstituted benzoic (μM) (μM) (μM) (μM) ID acid unit Iso, hypo Iso, hypoIso, hypo Iso, hypo 1a (ZT-1a)

1, 3 1, 1 3, 3 1, 3 1b

3, 3 3, 1 10, 10 3, 3 1c

3, 3 1, 3 10, 10 3, 3 1d

3, 3 1, 3 10, 10 3, 3 1e

— — — — 1f

3, 3 1, 3 10, 10 3, 3 1g

3, 3 1, 3 10, 10 3, 3 1h

3, 3 1, 3 10, 10 3, 3 1i

— — — — 1j

— — — — “—” showed no inhibition of phosphorylation of SPAK/NKCC1/KC22

The highest potency salicylic amides selected from the library werecompared with Closantel, STOCK1S-14279, and STOCK1S-50699 as SPAKinhibitors in a cellular context. NKCC1 and KCCs (KCC1-4) arephospho-substrates of SPAK kinase (de Los Heros et al. (2014) TheBiochemical journal 458, 559-573; Zhang et al. (2016) Scientific reports6, 35986). SPAK activity was monitored as NKCC1 Thr203/207/212phosphorylation, required for cotransporter activation (Vitari et al.(2005) The Biochemical journal 391, 17-24), and KCCs sites-1/2 (KCC2Thr906/1007 or KCC3 Thr991/1048) phosphorylation, required forcotransporter inhibition (de Los Heros et al. (2014) The Biochemicaljournal 458, 559-573; Rinehart et al. (2009) Cell 138, 525-536) (FIG.1B, FIG. 2, and FIG. 3). ZT-1a emerged as the most potent compound,exhibiting dose-dependent inhibition of NKCC1 p-Thr203/207/212 (up to72±5.2% inhibition at 1 μM; p<0.01; n=4) and KCCs sites-1/2phosphorylation (up to 65-77% inhibition at 3 μM; p<0.01; n=4) in HEK293cells (Table 1). Substantial dephosphorylation of SPAK Ser373 was alsoobserved at approximately 3-10 μM concentrations (representing 70±3.8%inhibition; p<0.01; n=3; FIG. 1B). Without wishing to be bound bytheory, these results confirm ZT-1a as a potent modulator ofSPAK-dependent CCC phosphorylation, in contrast to the existing SPAKkinase inhibitors Closantel, STOCK1S-50699 and STOCK1S-14279, which onlyat 10 μM significantly inhibited phosphorylation of KCCs site-1.

Referring to FIG. 1B, ZT-1a dose-dependently inhibited KCC2Thr906/Thr1007 phosphorylation in cells. HEK293 cells were transfectedwith a DNA construct encoding wild type N-terminally FLAG-tagged KCC2.36 hrs post-transfection, cells were exposed 30 min to either controlisotonic or hypotonic low [C1⁻] conditions, then treated in the sameconditions for an additional 30 min with the indicated inhibitors at theconcentrations noted. Cell lysates were subjected to SDS-PAGE andWestern blotting with the indicated antibodies. See FIG. 3 for Westernblot quantitation.

Referring to FIG. 2, concentration-response experiments testing effectsof Closantel analogs on phosphorylation of KCC2 Thr906/Thr1007 areshown. HEK293 cells were transfected with DNA construct encoding wildtype N-terminally FLAG-tagged KCC2. 36 h post-transfection, cells wereexposed 30 min to either control isotonic conditions or hypotonic lowCl⁻ conditions, then treated in the same conditions with the specifiedinhibitors (1b, 1c, 1f, 1d, 1h, and 1g, respectively) at the indicatedconcentrations for an additional 30 min. Lysates were subjected toSDS-PAGE and Western blotting with the indicated antibodies.

Referring to FIG. 3, HEK293 cells were transfected with the DNAconstruct encoding wild type N-terminally FLAG-tagged KCC2. 36 hpost-transfection, cells were exposed 30 min to either control isotonicor hypotonic low Cl⁻ conditions, then treated in the same conditionswith the specified inhibitors at the indicated concen-trations for anadditional 30 min. Lysates were subjected to SDS-PAGE and Westernblotting with the indicated antibodies. Closantel, STOCK1S-14279 andSTOCK1S-50699 were used as reference compounds. See FIG. 1B for Westernblots.

Next, it was tested whether kinase inhibition by ZT-1a reflectscompetition for ATP binding to kinase, as compared to the non-specificATP-competitive kinase inhibitor, staurosporine, which, in addition toinhibiting SPAK/OSR1 activity, binds to as many as 253 human proteinkinases. As shown in FIG. 4A, staurosporine IC₅₀ values increased in adose-proportional manner with increasing ATP concentrations, in contrastto the unchanged ZT-1a IC₅₀ values (FIG. 4B). Without wishing to bebound by theory, these results suggest that ZT-1a inhibits SPAK kinasein a non-ATP-competitive manner. The kinase selectivity of ZT-1a wasfurther assessed using standard radioisotopic enzymatic assays against apanel of 140 recombinant kinases (Dundee profiling, Table 2) (Bain etal. (2007) The Biochemical journal 408, 297-315). ZT-1a at aconcentration of 10 μM inhibited only MAPKAP-K2 activity 85±7% comparedto DMSO control, representing high kinase selectivity.

Referring to Table 2, ZT-1a was tested at 10 and 1 μM against a panel of140 recombinant kinases in an enzymatic activity-based assay performedby the International Centre for Kinase Profiling in the MRC ProteinPhosphorylation and Ubiquitylation Unit. Values represent % residualkinase activity (mean+/−SEM, n=3). Kinases labelled with an asterick*are inhibited ≥50% by ZT-1a.

TABLE 2 Concentration (μM) Kinases 10 1 ABL  68 ± 16 85 ± 4 AMPK (hum)108 ± 4  126 ± 4  ASK1 100 ± 11 101 ± 3  Aurora A 65 ± 1  73 ± 19 AuroraB  92 ± 15 119 ± 6  BRK 103 ± 2  116 ± 12 BRSK1  88 ± 22 108 ± 14 BRSK2104 ± 0  115 ± 14 BTK 73 ± 2 113 ± 1  CAMK1  71 ± 31 60 ± 5 CAMKKb 102 ±9  115 ± 10 CDK2-Cyclin A 125 ± 6  128 ± 12 CDK9-Cyclin T1 108 ± 1  104± 0  CHK1 109 ± 8  111 ± 11 CHK2 68 ± 6 106 ± 1  CK1γ2 124 ± 23 112 ± 12CK1δ 114 ± 9  116 ± 5  CK2  59 ± 16  72 ± 15 CLK2*  33 ± 45  68 ± 12 CSK106 ± 29 96 ± 8 DAPK1 159 ± 11 131 ± 8  DDR2 99 ± 8 111 ± 20 DYRK1A 103± 1  119 ± 7  DYRK2  97 ± 10 111 ± 15 DYRK3  56 ± 15 113 ± 6  EF2K 56 ±2 104 ± 13 EIF2AK3 100 ± 3  114 ± 2  EPH-A2  78 ± 11  89 ± 23 EPH-A4  90± 11  59 ± 33 EPH-B1  80 ± 19  77 ± 15 EPH-B2 81 ± 8 77 ± 2 EPH-B3 84 ±6 88 ± 6 EPH-B4 151 ± 33 100 ± 8  ERK1 109 ± 18 107 ± 16 ERK2 75 ± 3 111± 25 ERK5 114 ± 5  120 ± 9  ERK8 63 ± 3 104 ± 11 FGF-R1 101 ± 15 93 ± 6GCK 97 ± 3 112 ± 4  GSK3b*  38 ± 12 39 ± 4 HER4 84 ± 1 85 ± 8 HIPK1 90 ±4  98 ± 13 HIPK2 91 ± 4  90 ± 14 HIPK3 76 ± 2 84 ± 3 IGF-1R 123 ± 3  145± 2  IKKb 78 ± 3 101 ± 0  IKKe 59 ± 5  87 ± 13 IR  86 ± 13 105 ± 2 IRAK1 95 ± 7 128 ± 2  IRAK4 127 ± 11 127 ± 0  IRR 95 ± 4 105 ± 5  JAK2123 ± 8  125 ± 6  JNK1 110 ± 5  106 ± 4  JNK2 147 ± 51 102 ± 2  JNK3  60± 10  63 ± 15 Lck 127 ± 14 112 ± 13 LKB1 115 ± 11 107 ± 8  MAP4K3 88 ± 2134 ± 5  MAP4K5 76 ± 9  95 ± 28 MAPKAP-K2* 13 ± 7  91 ± 13 MAPKAP-K3 72± 8  96 ± 26 MARK1 91 ± 5 104 ± 16 MARK2  89 ± 15 103 ± 8  MARK3 68 ± 871 ± 6 MARK4 59 ± 9 68 ± 8 MEKK1  93 ± 16 109 ± 1  MELK 63 ± 3 130 ± 34MINK1 108 ± 6  107 ± 5  MKK1 114 ± 17 106 ± 2  MKK2  81 ± 27 126 ± 2 MKK6  82 ± 16 102 ± 12 MLK1  81 ± 21 94 ± 4 MLK3 114 ± 3  119 ± 11 MNK187 ± 2  78 ± 19 MNK2 105 ± 3  100 ± 7  MPSK1 105 ± 2  110 ± 6  MSK1 112± 14 114 ± 6  MST2 113 ± 2  115 ± 4  MST3 113 ± 15 109 ± 8  MST4 63 ± 0 63 ± 10 NEK2a 91 ± 6  85 ± 12 NEK6 108 ± 1  107 ± 6  NUAK1  61 ± 16 52± 7 OSR1 98 ± 3 106 ± 9  p38a MAPK 113 ± 2  128 ± 7  p38b MAPK 124 ± 0 124 ± 1  p38d MAPK 112 ± 2  95 ± 1 p38g MAPK 100 ± 6  100 ± 11 PAK2 93 ±8 110 ± 4  PAK4 51 ± 3  81 ± 16 PAK5 93 ± 4  93 ± 26 PAK6 75 ± 4 69 ± 2PDGFRA 80 ± 7  84 ± 11 PDK1  69 ± 17 92 ± 6 PHK 107 ± 2  97 ± 3 PIM1 101± 8  108 ± 19 PIM2 93 ± 3 93 ± 5 PIM3 99 ± 2 115 ± 12 PINK 59 ± 4 101 ±13 PKA* 50 ± 2  80 ± 11 PKBa 91 ± 4 104 ± 1  PKBb  51 ± 13  76 ± 14 PKCa83 ± 2 97 ± 4 PKCz 122 ± 28  80 ± 11 PKCγ 120 ± 8  133 ± 11 PKD1  79 ±10 89 ± 0 PLK1  93 ± 21 72 ± 1 PRAK* 41 ± 7  78 ± 13 PRK2 120 ± 7  118 ±4  RIPK2 120 ± 3  128 ± 5  ROCK2 101 ± 10 109 ± 6  RSK1 87 ± 4 95 ± 2RSK2 101 ± 3  116 ± 5  S6K1 81 ± 2 119 ± 10 SGK1 58 ± 5  73 ± 14 SIK2 76± 8 105 ± 16 SIK3 88 ± 0 104 ± 9  SmMLCK 98 ± 9 99 ± 3 Src  84 ± 13 107± 4  SRPK1  73 ± 20 104 ± 5  STK33 100 ± 5  114 ± 7  SYK 93 ± 1  93 ± 12TAK1 103 ± 5  117 ± 22 TAO1 104 ± 2  66 ± 2 TBK1 94 ± 3 119 ± 10 TESK1 77 ± 10 109 ± 3  TGFBR1 77 ± 5 63 ± 9 TIE2  73 ± 11 139 ± 74 TLK1 124 ±4  130 ± 13 TrkA 91 ± 7 108 ± 9  TSSK1 74 ± 6 99 ± 2 TTBK1 102 ± 2  119± 5  TTBK2  98 ± 34 101 ± 32 TTK 91 ± 5 104 ± 2  ULK1  89 ± 13  87 ± 10ULK2* 49 ± 9 110 ± 11 VEG-FR  88 ± 28 113 ± 10 WNK1 122 ± 35 125 ± 17YES1 98 ± 7 109 ± 3  ZAP70 97 ± 9 120 ± 1  Results are presented as thepercentage of kinase activity compareed with control incubations inwhich inhibitor was omitted. The results are means ± S.D. of triplicatedeterminations.

b. ZT-1a Disrupts SPAK Interaction with WNK but not with MO25α

Crystallographic analysis of the human OSR1 conserved carboxy-terminal(CCT) domain complexed to an RFXI motif-containing peptide derived fromWNK (Villa et al. (2007) EMBO reports 8, 839-845) has shown that thehighly conserved Leu473 CCT (mouse SPAK Leu502) forms criticalhydrophobic contacts with the Phe residue of the RFXI motif. In vitrofluorescence polarization studies have confirmed that the RFXImotif-containing WNK peptide binds to purified wild-type SPAK proteinwith 0.3 μM binding affinity, an interaction disrupted by STOCK1S-50699with IC₅₀ 2.51 μM) (FIG. 5A and FIG. 5B). Closantel and ZT-1a did notdisrupt binding between WNK4 and the SPAK CCT domain, suggesting theZT-1a binding site lies outside the CCT domain (FIG. 5A and FIG. 5B).Nonetheless, co-immunoprecipitation of WNK1 with SPAK from HEK293 celllysates was potently abolished by ZT-1a (FIG. 6). IC₅₀ values for ZT-1abinding were not significantly altered by addition of MO25α, whichactivates SPAK/OSR1 up to 100-fold and increases SPAK/OSR1-mediated invitro phosphorylation of all CCCs (de Los Heros et al. (2014) TheBiochemical journal 458, 559-573; Filippi et al. (2011) The EMBO journal30, 1730-1741) by 8-fold (FIG. 7A and FIG. 7B).

Referring to FIG. 5A, analysis of SPAK-WNK interaction by fluorescencepolarization is shown. Purified human SPAK 452-547(end) and human SPAK452-547 with L491A (equivalent to L502 in mouse) were dilutedappropriately and mixed at a 1:1 volume ratio with 20 nMLumino-Green-labelled WNK peptide (RFQV or AFQV) to the concentrationstated in the Figure (with the final peptide concentration consistent at10 nM), and fluorescent polarization measurements were made. Bindingcurves, assuming one-site-specific binding, were then generated withPrism6 using milli-polarization (mP) units.

Referring to FIG. 5B, under the assay conditions used in FIG. 5A,concentration-dependent decreases in fluorescence polarization weredetermined for STOCK1S-50699 (IC₅₀ 2.51 μM), Closantel (IC₅₀>1000 μM),and ZT-1a (IC₅₀ 325.5 μM).

Referring to FIG. 6, evidence that SPAK associates with WNK1, andinteraction is disrupted by SPAK CCT mutation is shown. Non-transfectedHEK293 cell lysates were incubated with the RFQV peptide(SEEGKPQLVGRFQVTSSK), AFQV peptide (SEEGKPQLVGAFQVTSSK), STOCK1S-50699,Closantel or ZT-1a for 30 min on ice and subjected to SPAK antibodyimmunoprecipitation. Immunoprecipitates were subjected to immunoblotprobed with antibody to total WNK1 and antibody to total SPAK.

Referring to FIG. 7A, the activation of SPAK kinase by MO25α is shown.SPAK was assayed in the absence or presence of five-fold molar excess ofwild-type MO25α.

Referring to FIG. 7B, kinetic data was determined for ZT-1a in theabsence or presence of five-fold molar excess of wild-type MO25α. Datapoints are the average of three determinations, and the error bars are+/−SEM. IC₅₀ results are 40.5 and 41.3 μM for ZT-1a in the respectiveabsence or presence of wild-type MO25α.

c. ZT-1a Reduces NKCC1 and KCC2 Phosphorylation at the CriticalSPAK-Regulated Phosphorylation Sites

To assess CCC phosphorylation in response to ZT-1a, HEK293 cells wereexposed for 30 min either to control isotonic conditions or to hypotoniclow [Cl⁻] (to activate SPAK/OSR1), then treated with increasing ZT-1aconcentrations for an additional 30 min. In a dose-dependent manner(1-10 ZT-1a markedly inhibited SPAK/OSR1 phosphorylation atSer373/Ser325 (i.e., the activating site phosphorylated by WNK1) andNKCC1 phosphorylation at Thr203/Thr207/Thr212 (SPAK/OSR1 target siteswhose phosphorylation is required for maximal transporter activity) inhypotonic low [Cl⁻] conditions as well as in control isotonic conditions(FIG. 1B and FIG. 3). These effects were paralleled by a similarsuppression of KCC2 Thr906/1007 phosphorylation, consistent withWNK-SPAK/OSR1-mediated phosphorylation of these residues (de Los Heroset al. (2014) The Biochemical journal 458, 559-573; Zhang et al. (2016)Scientific reports 6, 35986). Without wishing to be bound by theory,these results show that ZT-1a reduces the stimulatory phosphorylation ofNKCC1 at Thr203/Thr207/Thr212 and the inhibitory phosphorylation of KCCssites-1/2 phosphorylation.

d. ZT-1a Promotes KCC-Dependent Cellular Cl⁻ Extrusion

The potential clinical utility of specific KCC2 activators has led tointense development efforts (Gagnon et al. (2013) Nature medicine 19,1524-1528). The ability of ZT-1a to decrease inhibitory KCC2phosphorylation at Thr906/1007 prompted an assessment of ZT-1a's effecton KCC2 activity by measuring ⁸⁶Rb⁺ uptake in isotonic or hypotonic lowCl⁻ conditions (FIG. 8). Low KCC2 activity observed in wild-type (WT)cells was consistent with maximal KCC2 phosphorylation at Thr906/1007 inhypotonic low [Cl⁻] conditions or isotonic high [K⁺] (Kahle et al.(2013) Trends Neurosci 36, 726-737). In contrast, cells expressing KCC2double mutant Thr906A1a/Thr1007Ala, mimicking activatingdephosphorylation at these sites (Friedel et al. (2015) Sciencesignaling 8, ra65), exhibited 3.4-fold increased KCC2 activity comparedto WT KCC2 (p<0.01; n=3). ZT-1a activated WT KCC2>2.6-fold (p<0.01;n=3), whereas ZT-1a treatment failed only minimally increased (by 7±2%)activity of KCC2 double mutant Thr906A1a/Thr1007Ala (p>0.05; n=3; FIG.8). Without wishing to be bound by theory, these results show that ZT-1afacilitates KCC2-dependent Cl⁻ extrusion by decreasing itsSPAK-dependent inhibitory phosphorylation at Thr906/Thr1007.

Referring to FIG. 8, ⁸⁶Rb⁺ uptake assays in the presence of ZT-1a isshown. Specifically, referring to the Left Panel, HEK293 cells weretransfected with constructs encoding the indicated WT or mutantconstructs of N-terminally FLAG-tagged KCC2. 36 h post-transfectioncells were exposed for 30 min to either control isotonic conditions orhypotonic low [Cl⁻] conditions (to activate the SPAK/OSR1 pathway), thentreated in the same conditions for an additional 30 min with theindicated ZT-1a concentrations in the presence of 1 mM ouabain(Na⁺/K⁺-ATPase inhibitor) and 0.1 mM bumetanide (NKCC1 inhibitor). ⁸⁶Rb⁺uptake was allowed to proceed for 10 min and was then quantified byscintillation counting. ⁸⁶Rb⁺ uptake CPM's were normalized per mgprotein for each condition and plotted for both isotonic and hypotonicconditions. Referring to the Right Panel, HEK293 cells were transfectedwith constructs encoding wild type N-terminally FLAG epitope-taggedKCC2. 36 hrs post-transfection cells were exposed for 30 min to eithercontrol isotonic conditions or hypotonic low [Cl⁻] conditions (toactivate the SPAK/OSR1 pathway), then treated for an additional 30 minin the same conditions with the indicated concentrations of ZT-1a in thepresence of 1 mM ouabain and 0.1 mM bumetanide. 10 min ⁸⁶Rb⁺ uptakevalues were quantified by scintillation counting, normalized per mgprotein for each condition and plotted for both isotonic and hypotonicconditions. Statistical significance was determined at p<0.05 (bothpanels).

e. SPAK Inhibitory Effects of ZT-1a on CCC Phosphorylation are ShortActing and Reversible In Vivo.

To define ZT-1a efficacy in naive mice in vivo, phosphorylation of SPAK,NKCC1 and NCC in kidney and phosphorylation of SPAK, NKCC1 and KCC2 inbrain were examined, after intraperitoneal (i.p.) administration ofeither ZT-1a (10, 30, 50 and 100 mg/kg) or Closantel (50 mg/kg as areference). NCC phosphorylation (pThr46/50/55/60) and NKCC1phosphorylation (pThr203/207/212) was reduced ˜83-90±7.9% in naive WTkidneys 30 minutes after ZT-1a administration (50 mg/kg; p<0.01 n=3;FIG. 9B). In contrast, 60 minutes after administration of ZT-1a orClosantel, neither drug reduced KCC2 phosphorylation (pThr1007) or NKCC1phosphorylation (pThr203/207/212) in naïve brains (FIG. 9A). KCC2 Leu502is a residue required for high affinity recognition of the RFXI motif inSPAK upstream activator WNKs, as well as in its substrates NCC andKCC2/4 (Villa et al. (2007) EMBO reports 8, 839-845; Zhang et al. (2015)Human molecular genetics 24, 4545-4558). A ˜34±4.6% reduction in KCC2phosphorylation at pThr1007 was detected in brains from SPAK^(502A/502A)mice; p<0.01; n=3; FIG. 9A), a model of Gitelman Syndrome (Zhang et al.(2015) Human molecular genetics 24, 4545-4558). Thus, systemicallyadministered ZT-1a has low efficacy in naïve brains, indicatingapparently inefficient transport of ZT-1a across the blood-brain barrier(BBB), and likely reflecting in part its T_(1/2) of 1.8 hr in mice, withAUC of 2340 hr*ng/mL and % F of 2.2% (Table 3).

Referring to FIG. 9A and FIG. 9B, in vivo pharmacodynamic analysis ofZT-1a was performed. ZT-1a and Closantel were administered viasubcutaneous injection at the indicated doses. Brain (FIG. 9A) andkidney (FIG. 9B) tissues were collected, and endogenous proteins weresubjected to immunoblot probed with SPAK, NKCC1 and NCC phospho-specificantibodies.

Referring to Table 3, ZT-1a pharmacokinetics were determined following asingle 5 mg/kg intravenous (IV) dose and a single 10 mg/kg oral (PO)dose in ICR mice (N=3 at each time point). Blood samples were collectedat 0.08, 0.25, 0.5, 1, 2, 4, 8, 10 & 24 hr (IV) & 0.25, 0.5, 1, 2, 4, 8,10 & 24 hr (PO) post dose. Samples were subjected to drug assay byLC-MS/MS, and data were analyzed by WinNonlin V6.3. T_(max)=time ofmaximum plasma concentration, C_(max)=maximum plasma concentration,AUC=area under the curve (measure of exposure), T_(1/2)=half life,CL=plasma clearance, Vz=volume of distribution, MRT, mean residencetime; F=oral bioavailability.

TABLE 3 Dose t_(1/2) T_(max) C_(max) C₀ AUC_((0-t)) AUC_((0-∞)) Route(mg/kg) hr hr ng/mL ng/mL ng*hr/mL ng*hr/mL Plasma IV 5 1.8 0.08 691014900 2340 2350 PO 10 2.6 0.25 94 — 97.3 105 Vz Cl MRT_((0-∞)) F RouteL/kg mL/kg*min hr % Plasma IV 0.948 35.5 0.45 — PO — — 3.3 2.2

f. Intracerebroventricular (ICV) Delivery of ZT-1a NormalizesPathological CSF Hypersecretion by Decreasing Choroid Plexus CCCPhosphorylation

IVH-triggered TLR4 signaling stimulates CSF secretion >3.5-fold to causehemorrhagic hydrocephalus by increasing functional expression of pSPAKand pNKCC1 in the choroid plexus epithelium (CPe) (Karimy et al. (2017)Nature medicine 23, 997-1003). It was hypothesized thatintracerebroventricular (ICV) administration of ZT-1a, bypassing theBBB, might permit therapeutic efficacy by allowing ZT-1a access toSPAK-NKCC1 in the CPe. The effects of ICV ZT-1a delivery were tested onpSPAK, pNKCC1, pKCC4, in rat choroid plexus in the setting ofexperimental hemorrhagic hydrocephalus, as described (Karimy et al.(2017) Nature medicine 23, 997-1003)). (KCC2 is expressed in choroidplexus at very low or undetectable levels). ZT-1a reduced IVH-inducedexpression of pSPAK by 55±3.6%; p<0.01; n=3) and pNKCC1 by 69±4.3%;p<0.001; n=3; FIG. 10A and FIG. 10B). ZT-1a reduced post-IVH KCC4phosphorylation at Thr926 and Thr980 (corresponding to KCC2 Thr906 andThr1007) in CPe by ˜45-48±5.1%; p<0.001; n=3; FIG. 10A and FIG. 10B).Consistent with these results, ZT-1a treatment for 48 hr at 10 mmoldecreased post-IVH CSF hypersecretion by ˜57±6.2% (p<0.01), in contrastto the lack of effect of DMSO vehicle treatment (p>0.05) (FIG. 10B).Without wishing to be bound by theory, these data suggest ICVadministration of ZT-1a can effectively modulate pathological CSFsecretion by decreasing SPAK-CCC phosphorylation.

Referring to FIG. 10A, the effect of ZT-1a (ICV; 10 mmol) on IVH-inducedphosphorylation of SPAK/OSR1, NKCC1, KCC2, and KCC4 in CPE, as measuredin control rats (CTL) and in rats 48 hrs post-IVH treated with ZT-1a orvehicle (n=3 for all groups) is shown. CPE lysates were harvested andsubjected to immunoprecipitation (IB) and immunoblot (IB) with theindicated antibodies. D, dimeric KCC2; M, monomeric KCC2. Molecular massis indicated in kDa.

Referring to FIG. 10B, the effect of SPAK inhibition by ZT-1a onIVH-induced CSF hypersecretion by CPE is shown. The rate of CSFproduction (μL/min) is presented as means±SEM. **, p<0.01 vs. control;#, p<0.01 vs. IVH but not vs. controls (p>0.05); one-way ANOVA.Quantitation of actin-normalized choroid plexus ion transporterphosphorylation is presented in control rats (CTL) and rats 48 hrs afterexperimental IVH in the presence or absence of ZT-1a or vehicle (n=3 forall groups). *, p<0.05 versus control rats, one-way ANOVA.

g. Post-Stroke Administration of ZT-1a Reduces Ischemic Cerebral Edema

Ischemic stroke is associated with significant up-regulation of SPAK andNKCC1 phosphorylation in peri-infarct cortex, striatum and corpuscallosum (Begum et al. (2015) Stroke 46, 1956-1965). Genetic inhibitionof either SPAK or NKCC1 decreases ischemic cerebral edema and improvesneurological outcomes (Zhang et al. (2017) Neurochem Int 111, 23-31;Zhao et al. (2017) Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism 37, 550-563). The efficacy of ZT-1a on development ofcerebral infarct and associated cerebral edema in a mouse model ofischemic stroke was evaluated (Begum et al. (2015) Stroke 46, 1956-1965;Zhao et al. (2017) Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism 37, 550-563). Post-stroke administration of ZT-1a (5 mg/kg)at 3 hr reperfusion decreased infarct volume by ˜35% (from 78.0±20.0 mm³in the vehicle-control group to 50.4±27.0 mm³, p<0.05, FIG. 11A and FIG.11B). Moreover, ZT-1a treatment at either 2.5 or 5 mg/kg reducedcerebral hemispheric swelling by ˜36 to 50% as compared tovehicle-control values (p<0.05, FIG. 11B). ZT-1a did not affect regionalcerebral blood flow (rCBF) within the 24 hr post-stroke period (FIG.12A-C). These results show that the novel SPAK inhibitor ZT-1a iseffective in reducing ischemic brain infarct and edema.

Referring to FIG. 11A, an experimental design of ischemic stroke studyis shown. Vehicle or ZT-1a is administered at 3 hrs and 8 hrs post-MCAO.

Referring to FIG. 11B, representative images and quantification ofinfarct volume and hemispheric swelling in TTC-stained coronal sectionsof mouse brains 24 hrs post-MCAO are shown. Vehicle (DMSO, 2 ml/kg) orZT-1a (2.5 or 5.0 mg/kg) were administered with an initial half dose at3 hrs and the second half dose at 8 hr reperfusion via intra-peritonealinjection (i.p.). Data are mean±SD, n=6-12 per group (male). * p<0.05.

Referring to FIG. 12A, representative two-dimensional laser specklecontrast images of rCBF are shown. Vehicle (DMSO) or ZT-1a (2.5mg/kg/dose) was administered at 3 hr and again at 8 hr post-reperfusionafter MCAO, respectively. PseudocolorScale bar indicates relative signalintensity.

Referring to FIG. 12B, no changes of rCBF were detected in thenon-ischemic contralateral (CL) hemispheres of vehicle-control orZT-1a-treated mice. Data are mean±SD, n=3.

Referring to FIG. 12C, similar reduction and recovery of rCBF in theischemic ipsilateral hemispheres (IL) were detected in the vehicle- orZT-1a-treated ischemic mice. Data are mean±SD, n=3.

h. ZT-1a Improves Neurological Function after Ischemic Stroke

The impact of ZT-1a on progression of sensorimotor function deficits inthe mouse model of ischemic stroke was further evaluated using an arrayof neurobehavioral tests. The vehicle-control mice developed persistentsevere neurological deficits at days 0-7 after stroke, as reflected inelevated neurological scores of 2.5-2.9 (FIG. 13). ZT-1a-treated miceexhibited a progressive decrease in neurological deficit scores betweenday 1 (2.0±0.2) and day 7 (1.4±0.2, p<0.05). In the corner test (Zhao etal. (2017) Journal of cerebral blood flow and metabolism: officialjournal of the International Society of Cerebral Blood Flow andMetabolism 37, 550-563) evaluating post-ischemic sensory and motordeficits, vehicle-control mice exhibited behavioral asymmetries 1 dayafter stroke that persisted for 7 days (FIG. 13), whereas ZT-1a-treatedmice showed reduced unidirectional turn preference and absence ofasymmetries by day 7 post-stroke. In the adhesive contact and removaltests that evaluate fine sensorimotor function deficits (Begum et al.(2018) Glia 66, 126-144), ZT-1a-treated mice displayed a trend to fastercontact response than in vehicle-control mice (although short ofstatistical significance; p=0.10), and significantly improved motorfunction (p<0.05). These data show that post-stroke treatment with ZT-1ain mice significantly improved neurological functional recovery.

Referring to FIG. 13, neurological deficit scores, corner tests, andadhesive tape contact and adhesive tape removal tests of mice treatedwith vehicle or ZT-1a 1 day before tMCAO (˜1) and at days 0, 1, 3, 5,and 7 post-tMCAO are shown. Vehicle (DMSO, 2 ml/kg) or ZT-1a (5.0 mg/kg)were administered as described in B. Data are means±SEM, n=6 for eachgroup (male 3, female 3). * p<0.05 vs. vehicle.

i. ZT-1a Inhibits Stroke-Induced SPAK-NKCC1 Phospho-Activation inIschemic Brains.

Next, the effect of ZT-1a on SPAK/OSR1, NKCC1, and KCC3 phosphorylationin ischemic mouse brains was tested. Ischemic stroke increasedphosphorylation of pSPAK (pSer373)/pOSR1 (pSer325) ˜1.5 fold (p<0.05),pNKCC1 (pThr203/207/212) ˜1.6 fold (p<0.05), and pKCC3 pThr991 (p<0.05)and pThr1048 ˜1.3-fold (both p<0.05) in membrane fractions from theipsilateral (IL) cortical hemisphere at 24 hrs reperfusion invehicle-control treated mice, without change in levels of correspondingtotal proteins (FIG. 14A and FIG. 14B). Post-stroke administration ofZT-1a in mice prevented ischemia-induced increases of pSPAK/pOSR1,pNKCC1, and pKCC3 without affecting corresponding total proteinexpression (p<0.05) (FIG. 14A and FIG. 14B). Without wishing to be boundby theory, these results indicate that ZT-1a inhibits SPAK-dependentphospho-stimulation of NKCC1 and inactivation of KCC3 in ischemicbrains.

Referring to FIG. 14A, representative immunoblots (IB) ofphospho-SPAK/OSR1 (pSPAK/pOSR1), phospho-KCC3 and phospho-NKCC1 (pNKCC1)in mouse brains studied 24 hrs post-reperfusion after ischemic strokeare shown. Membrane protein fractions were prepared from contralateral(CL) and ipsilateral (IL) cerebral hemispheres. Vehicle (Veh, DMSO) orZT-1a (5 mg/kg) was administered as described in FIG. 11A, FIG. 11B, andFIG. 13. Na⁺-K⁺ ATPase α-subunit and GAPDH served, respectively, asloading controls for membrane and cytosol fractions.

Referring to FIG. 14B, densitometry analyses of immunoblots (similar tothose in panel A) of pSPAK/pOSR1, pNKCC1, tSPAK/tOSR1, and tNKCC1 inmouse brains studied 24 hrs reperfusion after tMCAO are shown. Data aremeans±SEM, n=5 per group (male 3, female 2). *p<0.05.

j. ZT-1a-Treated Mice Exhibited Persistent Protection of Grey and WhiteMatter after Ischemic Stroke.

Ex vivo MRI studies of brains from vehicle-control and ZT-1a-treatedmice at 7 days post-stroke were also conducted. T2-weighted MRI analysisfurther confirmed that ZT-1a treatment reduced stroke-induced lesionvolume by ˜40% and brain atrophy (hemisphere shrinkage) by ˜41% (p<0.05;FIG. 15A). To assess the effect of ZT-1a on stroke-mediated white matterinjury, we analyzed fractional anisotropy (FA), axial diffusivity (AD),radial diffusivity (RD) and mean diffusivity (MD) and directionallyencoded color (DEC) maps of the corpus callosum (CC) and externalcapsule (EC) in vehicle-control and ZT-1a-treated brains. Representativeimages of DEC and FA maps (FIG. 15B) reveal intact CC and EC (arrows) inthe CL hemisphere and injured EC in the IL hemisphere (arrowheads).Whereas FA values were reduced in the ipsilateral EC tract of thevehicle-control group, indicating loss of white matter integrity insubacute stroke brains. In contrast, EC tract FA values in the ILhemisphere of ZT-1a-treated mice did not change (p>0.05), reflectingpreserved EC white matter microstructure. These results further suggestthat post-stroke treatment with ZT-1a provides robust neuroprotection ofboth gray and white matter in ischemic brains.

Referring to FIG. 15A, representative T2WI images and quantitativeanalyses of lesion volume and atrophy (shrinkage) of ex vivo brains fromvehicle (Veh) control and ZT-1a-treated mice at 7 days post-MCAO areshown. Vehicle (DMSO 2 ml/kg) or ZT-1a (5.0 mg/kg) was administered i.p.in divided doses at 3 and 8 hrs after reperfusion. Data are means±SEM,n=6 per group (male 3, female 3); *p<0.05.

Referring to FIG. 15B, representative images of directionally encodedcolor (DEC) and fractional anisotropy (FA) maps are shown. Arrow: EC(external capsule); double arrowhead: damaged EC; CC: corpus callosum.Bar graphs display quantitation of mean values of FA, mean diffusivity(MD), axial diffusivity (AD) and radial diffusivity (RD) of white mattertissues (CC or EC) of ex vivo brain from vehicle control (Veh) andZT-1a-treated mice at 7 days post-MCAO. Same cohort as A. Data aremeans±SEM, n=6 per group (male 3, female 3); *p<0.05.

k. ZT-1a is Superior to Closantel and to WNK463 in Reducing IschemicBrain Injury in Mice.

Post-stroke neuroprotective efficacy of Closantel and the pan-WNK-kinaseinhibitor WNK463 were compared. Administration of Closantel (1.0 or 2.5mg/kg) at 3 hr and at 8 hr reperfusion dose-dependently reduced infarctvolume and hemispheric swelling (p<0.05, FIG. 16A-C), whereas the 0.1mg/kg dose of Closantel was ineffective. Closantel, like ZT-1a, had noeffects on rCBF (FIG. 12A-C and FIG. 17A-C). In contrast, WNK463treatment (2.5 mg/kg) reduced neither infarct volume nor hemisphericswelling at 24 hr reperfusion (FIG. 16A-C). These data displaysuperiority of ZT-1a to WNK463 and Closantel for in vivo inhibition ofthe WNK-SPAK-CCC pathway in the mouse model of ischemic brain injury.

5. Novel SPAK Kinase Inhibitor ZT 1a is Neuroprotective in Mouse Modelof Malignant Ischemic Stroke

In sum, a potent and selective inhibitor of SPAK kinase, ZT-1a[5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide]has been discovered and characterized through a “scaffold-hybrid”strategy. ZT-1a inhibits stimulatory phosphorylation of SPAK Ser373 andNKCC1 Thr203/207/212, and inhibitory phosphorylation of the KCCs (e.g.,KCC3 Thr991/Thr1048) at a concentration of 3 μM in HEK293 cells toeffectively promote cellular Cl⁻ extrusion. ICV delivery of ZT-1a inrats normalizes CSF hypersecretion in experimental hemorrhagichydrocephalus by decreasing inflammation-dependent phosphorylation ofSPAK, NKCC1, and KCC4. Systemic administration of ZT-1a in mice afterischemic stroke attenuates cerebral edema and improves neurologicaloutcomes by reducing ischemia-induced phosphorylation of NKCC1 and KCC3.Without wishing to be bound by theory, these data suggest ZT-1a is anovel effective kinase-cotransporter modulator that restores brain waterhomeostasis and improves neurological function in vivo.

NKCC1 is potently inhibited by the commonly used loop diureticsbumetanide and furosemide. Furosemide has poor blood-brain barrier (BBB)penetration and significantly lowers NKCC1 affinity than bumetanide(Donovan et al. (2016) European journal of pharmacology 770, 117-125;Fischer et al. (1998) J Membrane Biol 165, 201-211; Puskarjov et al.(2014) Epilepsia 55, 806-818). Multiple studies in both animal modelsand humans suggest bumetanide is effective in reducing Cl⁻ influx andrestoring GABAergic inhibition (Dzhala et al. (2010) The Journal ofneuroscience: the official journal of the Society for Neuroscience 30,11745-1176; Kahle et al. (2009) Journal of child neurology 24, 572-576;Liu et al. (2012) Pediatric research 71, 559-565; Mazarati et al. (2009)Epilepsia 50, 2117-2122). However, bumetanide exhibits poor CNSpenetration due to its significant binding to serum albumin, and itsinhibition of the renal-specific isoform NKCC2 causes potent,use-limiting diuresis, an off-target for treating brain disorders. Inaddition, bumetanide usage in neonates has raised concerns that alteringpremature E_(GABA) depolarization by bumetanide may inhibit normalneurodevelopment (Wang and Kriegstein (2011) Cerebral cortex 21,574-587). Alternatively, studies have demonstrated the potential of KCC2activators as effective pharmacotherapies for disorders of GABAergicdisinhibition (Austin and Delpire (2011) Anesth Analg 113, 1509-1515).CLP257 was reported as a direct KCC2 activator (Gagnon et al. (2013)Cell physiology 304, C693-714); however, these results have recentlybeen challenged (Cardarelli et al. (2017) Nature medicine 23,1394-1396).

Problems associated with directly targeting NKCC or the KCCs werecircumvented by focusing on the upstream SPAK kinase that reciprocallystimulates NKCC1 and inhibits KCCs through phosphorylation at a sharedmotif. The strategy of targeting the ATP-binding site of SPAK/OSR1raises concern regarding the ability to develop sufficiently selectiveinhibitors that do not suppress other kinases. The introduction ofSTOCK1S-50699 and STOCK2S-26016 (Mori et al. (2013) The Biochemicaljournal 455, 339-345) has highlighted the possibility of developingadditional inhibitors of SPAK signaling which target the CCT domainrather than the kinase domain. In vitro studies, however, demonstratedthat only STOCK1S-50699, but not STOCK2S-26016, suppressedphosphorylation of SPAK/OSR1 and NKCC1 induced by hypotonic low [Cl⁻](de Los Heros et al. (2014) The Biochemical journal 458, 559-573), andfurther showed unfavorable in vivo pharmacokinetics of STOCK1S-50699(data not shown). The anti-parasitic drug Closantel, widely used inlivestock (Swan (1999) J S Afr Vet Assoc 70, 61-70), emerged as thefirst candidate drug for in vivo pharmacological inhibition of SPAK(Alamri et al. (2017) ChemMedChem 12, 639-645; Kikuchi et al. (2015)Journal of the American Society of Nephrology: JASN 26, 1525-1536), butcontraindicated in humans (Tabatabaei et al. (2016) BMC Ophthalmol 16,207),

The ATP-independence of SPAK inhibition by Closantel and STOCK1S-14279has introduced the possibility of developing inhibitors of WNK signalingby binding to constitutively active or WNK-insensitive (T233E) SPAK(Kikuchi et al. (2015) Journal of the American Society of Nephrology:JASN 26, 1525-1536). Through “scaffold-hybrid” strategy (Deng et al.(2011) Nature chemical biology 7, 203-205; Deng et al. (2013) Eur J MedChem 70, 758-767), a new focused chemical library derived from these twoATP-insensitive inhibitors was designed and synthesized (FIG. 1A). ZT-1awas screened and characterized as one of the best SPAK inhibitorcompounds (Table 1) from a chemical library of 300 compounds, throughfluorescence polarization assays, in vitro SPAK kinase assays, andcell-based assays. ZT-1a was more potent as a general CCC inhibitor thanthe previously existing SPAK inhibitors Closantel (Kikuchi (2015) NihonJinzo Gakkai shi 57, 1319-1322), Rafoxanide ((Alamri et al. (2017)ChemMedChem 12, 639-645), STOCK1S-14279 (Kikuchi (2015) Nihon JinzoGakkai shi 57, 1319-1322), and STOCK1S-50699 (Mori et al. (2013) TheBiochemical journal 455, 339-345), substantially inhibitedphosphorylation of SPAK Ser373, NKCC1 Thr203/207/212, KCC2 Thr906 andThr1007 (KCC4 Thr926 and Thr980) at a concentration of 3 μM in HEK293cells, and effectively promoted cell Cl extrusion.

In a mouse study, post-stroke administration of Closantel reducedischemic cerebral infarction and swelling in a dose-dependent manner.However, adverse effects in humans at higher doses (˜25 mg/kg),including weakness, visual impairment and blindness (Essabar et al.(2014) Asia Pacific Journal of Medical Toxicology 3, 173-175; Tabatabaeiet al. (2016) BMC Ophthalmol 16, 207) deterred further investigation ofClosantel in the setting of stroke or hydrocephalus. The pan-WNK-kinaseinhibitor WNK463 inhibits all four WNK kinases in the nanomolar range invitro and was developed as an anti-hypertensive drug (Yamada et al.(2016) Nature chemical biology 12, 896-898). Oral administration of 1-10mg/kg WNK463 reduced blood pressure and regulated body fluid andelectrolyte homeostasis in normotensive and hypertensive rodent models(Yamada et al. (2016) Nature chemical biology 12, 896-898). However,WNK463 administered to mice at 2.5 mg/kg dose showed no neuroprotectiveeffects, instead inducing ataxia and breathing difficulties which aresimilar to the previous report at 1-10 mg/kg doses (Yamada et al. (2016)Nature chemical biology 12, 896-898). The causes for these adverseeffects remain unclear, but they, too, discouraged efficacy testing ofWNK463 at higher doses.

Post-stroke administration of ZT-1a attenuated stroke-associatedinfarction and cerebral edema and rapidly improved neurological functionin post-ischemic mice, results corroborated by brain MRI. T2-weightedMRI of ex vivo brains from ZT-1a-treated mice showed smaller lesionvolumes, and reduced brain atrophy at 7 days post-stroke. DTI dataanalysis revealed that ZT-1a significantly reduced subacute brain whitematter injury after ischemic stroke. ZT-1a thus providesneuroprotection, at least in part, by directly inhibiting SPAK kinaseactivity and SPAK-mediated phospho-activation of NKCC1 and inactivationof KCC3 in ischemic brains. ZT-1a reduced ischemia-induced elevations ofpSPAK and pNKCC1 by 55-65%, and elevations of pKCC3 by 30% compared tovehicle-control mice. The findings that ZT-1a concurrently inhibitedphosphorylation of SPAK, KCC3 and NKCC1 are consistent with thisinterpretation, i.e., ZT-1a-mediated allosteric inhibition of SPAKkinase activity is likely through binding to the secondary pocket of theCCT domain, preventing SPAK binding and activation by upstream WNKkinases. A similar dual mechanism of WNK-SPAK inhibitor STOCK1S-50669has been reported in cultured cells (Alamri et al. (2017) ChemMedChem12, 639-645; Mori et al. (2013) The Biochemical journal 455, 339-345).

Plasma half-life of ZT-1a is only ˜1.8 hrs in normal naïve mice, andZT-1a apparently fails to penetrate the healthy BBB. However, ZT-1aappeared to more effectively enter the ischemic brain across its leakyBBB in exerting its neuroprotective effects. Ischemic stroke injuryincreases permeability and disrupts BBB tight junctions as early as at30 minutes of reperfusion, (Sandoval and Witt (2008) Neurobiol Dis 32,200-219; Shi et al. (2016) Nat Commun 7, 10523), which facilitates brainaccess for small molecule drugs (Won et al. (2011) Exp Mol Med 43,121-128). ZT-1a was administered with the initial dose at 3 hrs poststroke, a time is comparable to effective treatment windows for otherpotential neuroprotective candidates, such as glibenclamide, humanalbumin, and minocycline (at 2.5-4.0 hrs post-stroke) (Belayev et al.(2001) Stroke 32, 553-560; Simard et al. (2009) Lancet Neurol 6,258-268; Yrjanheikki et al. (1999) Proc Natl Acad Sci USA 96,13496-13500). ZT-1a is superior to the anti-parasitic drug Closantel andthe novel pan-WNK inhibitor WNK463 in reducing ischemic brain damage.Future investigations are needed for examining bioavailability andpharmacokinetics of ZT-1a in ischemic brains and optimizing treatmentprotocols.

The CPe secretes higher volumes of fluid (CSF) per cell than any otherepithelium (500 ml/day). NKCC1 expressed in the apical CPe contributesapproximately half of the CSF production via its unusual outwardtransport direction and its unique ability to directly couple watertransport to ion translocation (Steffensen et al. (2018) Nat Commun 9,2167). In a rat model of hemorrhagic hydrocephalus, intraventricularhemorrhage causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependentinflammatory response in CPe associated with ˜3-fold increasedbumetanide-sensitive CSF secretion (Karimy et al. (2017) Nature medicine23, 997-1003). IVH-induced hypersecretion of CSF is mediated byTLR4-dependent activation of SPAK, which binds, phosphorylates, andstimulates NKCC1 at the CPe apical membrane (Karimy et al. (2017) Naturemedicine 23, 997-1003). Genetic depletion of TLR4 or SPAK normalizeshyperactive CSF secretion rates and reduces posthemorrhagichydrocephalus symptoms by reducing NKCC1 phosphorylation. Here, it wasshown that ICV administration of ZT-1a restores CSF secretion rates tobasal levels after IVH and antagonizes inflammation-inducedphosphorylation of SPAK, NKCC1, and KCC4 in CPe. Without wishing to bebound by theory, this suggests that ZT-1a could be a novelpharmacological treatment for hydrocephalus, currently treatable only bythe highly morbid surgical approaches of endoscopy or shunting. Furtherwork will be required to assess the therapeutic potential of ZT-1a inother pre-clincal models of hydrocephalus.

In sum, the novel drug, ZT-1a, that potently and selectively inhibitsSPAK kinase, the master regulator of CCCs, was developed. ZT-1a promotescellular Cl⁻ extrusion by simultaneous reduction of SPAK-dependent NKCC1stimulatory phosphorylation and KCC inhibitory phosphorylation.Intracerebroventricular delivery of ZT-1a, by decreasinginflammation-induced NKCC1/KCC4 phosphorylation in the choroid plexus,normalizes CSF hypersecretion in hemorrhagic hydrocephalus. Systemicallyadministered ZT-1a, by reducing ischemia-induced NKCC1/KCC3phosphorylation, attenuates cerebral edema, protects against braindamage, and improves neurological outcomes after ischemic stroke.Without wishing to be bound by theory, these results suggest ZT-1a is aneffective kinase-cotransporter modulator with therapeutic potential fordisorders of impaired brain water homeostasis.

6. Therapeutic Application of ZT-1a and Derivatives Thereof for BrainDisorders

a. ZT-1a Penetrates Ischemic Brain of Normotensive Mice

Brain penetration of ZT-1a was examined in normotensive mice afterischemic stroke due to leaky BBB. ZT-1a was administered by a singleinjection of 5 mg/kg ZT-1a (i.p.) in normotensive sham-control mice orin the mice at 3 hrs after tMCAO. At 2 hrs post-injection, there was nosignificant difference in the plasma bioavailability of ZT-1a in shamcontrol mice and ischemic stroke mice (p=0.29; n=10-11). However, brainconcentrations of ZT-1a were significantly higher (˜1.8-fold; p=0.006;n=10-11) in the stroke mice than that of sham-controls. These studiessuggest that ischemic stroke facilitates ZT-1a penetration into braintissue, probably via the disrupted blood-brain barrier (BBB).

b. Both Male and Female Ang II-Mediated Hypertensive Mice are Responsiveto SPAK Inhibitor ZT-1a after pdMCAO.

Ang II infusion (osmotic minipump at 1000 ng/kg/min, s.c.) for 14 dayssignificantly elevated arterial BP in male mice. In contrast, 14-dayinfusion of Ang II in female mice did not increase systemic BP, which isconsistent with published reports on estrogen-mediated effects (Xue etal. (2005) Am J Physiol Heart Circ Physiol. 288:H2177-2184). These micewere randomly subjected to pdMCAO and post-stroke administration of DMSOvehicle (Veh) control or the novel SPAK inhibitor ZT-1a (2.5 mg/kg,i.p., with an initial dose at 3 hrs and the second dose at 8 hrs afterpdMCAO). Ang II-infused mice (both male and female) exhibitedsignificantly larger infarct volume and hemispheric swelling at 24 hrsafter pdMCAO, compared to saline-infused normotensive controls.Post-stroke administration of the novel SPAK inhibitor ZT-1a decreasedinfarct volume (˜50%) and hemispheric swelling (˜50%) in the AngII-infused mice (male or female) compared to Veh control.

Referring to FIG. 18A, Angotension II (Ang II) infusion (osmoticminipump at 1000 ng/kg/min, s.c.) for 14 days in both male or femalemice significantly elevated arterial BP in male mice. In contrast,14-day infusion of Ang II in female mice did not increase systemic BP(FIG. 18B), which is consistent with published reports onestrogen-mediated effects. These mice were randomly subjected to pdMCAOand post-stroke administration of DMSO vehicle (Veh) control or thenovel SPAK inhibitor ZT-1a (2.5 mg/kg, i.p., with an initial dose at 3hrs and the second dose at 8 hrs after pdMCAO).

Referring to FIG. 18C-E, Ang II-infused mice (both male and female)exhibited significantly larger infarct volume and hemispheric swellingat 24 hrs after pdMCAO, compared to saline-infused normotensivecontrols. Post-stroke administration of the novel SPAK inhibitor ZT-1adecreased infarct volume (˜50%) and hemispheric swelling (˜50%) in theAng II-infused mice (male or female, FIG. 18C-E), compared to Vehcontrol.

These findings demonstrate that female mice are equally sensitive toZT-1a treatment, despite the absence of elevated BP. Moreover, withoutwishing to be bound by theory, these findings further support the viewthat Ang II-mediated activation of WNK-SPAK-NKCC1 signaling occurs inthe CNS of both male and female mice, and ZT-1a effects are via blockingthe WNK-SPAK-NKCC1 complex in ischemic brains, unlikely through reducingsystemic BP.

c. Post-Stroke Administration of SPAK Inhibitor ZT-1a ImprovesNeurological Deficits in the Ang II-Mediated Hypertensive Mice afterpdMCAO.

Sensory-motor function was assessed in the Ang II-mediated HTN mice(male) at 1-14 days post-pdMCAO (FIG. 19A). Ang II-mediated HTN mice(Ang II alone, or Ang II+Veh treatment) exhibited worsened neurologicalfunction following pdMCAO, compared to the saline-infused normotensivecontrol group, in foot fault test (FIG. 19B), cylinder test (FIG. 19C),and adhesive tape removal test (FIG. 19D). In contrast, ZT-1a-treatedhypertensive mice displayed less foot faults, less unilateral turns andbetter performance in adhesive tape removal from the injured paws (FIG.19B-D).

To determine whether SPAK inhibitor ZT-1a treatment affected BP,systemic BP was measured in ZT-1a-treated mice after pdMCAO. FIG. 19Eshows that ischemic stroke significantly lowered BP in mice at 1 daypdMCAO. But, Veh- and ZT-1a-treated mice displayed similar systolic anddiastolic BP at 1 or 14 days after pdMCAO.

These findings suggest again that neuroprotective effects conferred byZT-1a in hypertensive mice following pdMCAO is BP-independent. Thesedata clearly show that Ang II-mediated HTN mice exhibited worsenedischemic infarction and neurological deficits after pdMCAO. Post-strokeinhibition of SPAK kinase with a novel SPAK inhibitor ZT-1a providedrobust neuroprotection against ischemic stroke-induced brain damage andaccelerated neurological recovery.

Cylinder test: Mice are placed in a transparent cylinder (9 cm indiameter and 15 cm in height) for 10 min and all the forelimb movementsof the mice will be recorded. Forepaw (left/right/both) usage on initialcontact against the cylinder wall after rearing and during lateralexploration will be recorded. Prior to ischemic stroke, animals wereplaced in the cylinder for 5 min to establish a baseline symmetryprofile.

Foot fault test: Each mouse will be placed on a stainless-steel gridfloor (20 cm×40 cm with a mesh size of 4 cm²) elevated 1 m above thefloor. The animals will first be habituated to the grid floor for 1 minand then tested for three 1-min trials. Data will be expressed as thenumber of foot fault errors made by the forelimbs contralateral to theinjury hemisphere as a percentage of total steps.

Adhesive contact and removal tests: A piece of adhesive tape (4 mm×3 mm)will be attached to the contralateral forepaw with equal pressure by theexperimenter in each trial. The time to make first contact with the tapeand the time to remove the tape will be recorded as the contact time andthe removal time, respectively. Each trial ends after the adhesive tapeis removed or after 2 min elapse.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otheraspects of the invention will be apparent to those skilled in the artfrom consideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

What is claimed is:
 1. A method for treating a hypoxic brain injury in asubject, the method comprising the step of administering to the subjectan effective amount of at least one compound selected from:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the compound is selected from:


3. The method of claim 1, wherein the compound is selected from:


4. The method of claim 1, wherein the compound is:


5. The method of claim 1, wherein the subject is a mammal.
 6. The methodof claim 1, wherein the subject is a human.
 7. The method of claim 1,wherein the subject has been diagnosed with a need for treatment of ahypoxic brain injury prior to the administering step.
 8. The method ofclaim 1, further comprising the step of identifying a subject in need oftreatment of a hypoxic brain injury.
 9. The method of claim 1, whereinthe effective amount is a therapeutically effective amount.
 10. Themethod of claim 1, wherein the effective amount is a prophylacticallyeffective amount.
 11. The method of claim 1, wherein the hypoxic braininjury is associated with dysregulation of SPAK kinase.
 12. The methodof claim 1, wherein the hypoxic brain injury is due to traumatic braininjury, ischemic stroke, carbon monoxide poisoning, drowning, choking,suffocating, or cardiac arrest.
 13. The method of claim 1, wherein thehypoxic brain injury is due to ischemic stroke.
 14. A method fortreating a hypoxic brain injury associated with dysregulation of SPAKkinase in a subject, the method comprising the step of administering tothe subject an effective amount of a compound having a structure:

or a pharmaceutically acceptable salt thereof.
 15. The method of claim14, wherein the subject is a mammal.
 16. The method of claim 14, whereinthe subject is a human.
 17. The method of claim 14, wherein theeffective amount is a therapeutically effective amount.
 18. The methodof claim 14, wherein the effective amount is a prophylacticallyeffective amount.
 19. The method of claim 14, wherein the hypoxic braininjury is due to traumatic brain injury, ischemic stroke, carbonmonoxide poisoning, drowning, choking, suffocating, or cardiac arrest.20. The method of claim 14, wherein the hypoxic brain injury is due toischemic stroke.